Protein synthetic patterns of tissues in the early chick embryo

Development ◽  
1985 ◽  
Vol 85 (1) ◽  
pp. 65-80
Author(s):  
Robin H. Lovell-Badge ◽  
Martin J. Evans ◽  
Ruth Bellairs
Keyword(s):  
Chick Embryo ◽  
Lower Layer ◽  
Polyacrylamide Gel Electrophoresis ◽  
The Other ◽  
Cell Type ◽  
Lateral Plate ◽  
Specific Expression ◽  
Mesodermal Cell ◽  
Stage 4

Tissues dissected from early chick embryos were labelled in vitro with [35S]methionine, and their patterns of polypeptide synthesis investigated using the technique of two-dimensional (2–D) polyacrylamide gel electrophoresis. Apart from providing a preliminary description of the molecular changes associated with the processes of gastrulation and segmentation in the chick embryo, this study has revealed a number of polypeptides that may be useful as markers of cell type or function. The protein synthetic patterns of hypoblast from early and late gastrulae (stages 2 and 4, respectively: Hamburger & Hamilton, 1951) and of definitive endoblast and junctional endoblast from late gastrulae all resemble one another closely, but differ markedly from that of the epiblast at either stage. The lower layer tissues are characterized by the presence of eleven polypeptides that are largely absent from the epiblast. These findings are discussed with reference to current theories on the origins of the lower layer tissues. Comparisons between the 2-D patterns for tissues dissected from gastrulae and from embryos undergoing segmentation (stage 12) have revealed ten polypeptides showing stage-specific rather than tissue-specific expression. Apart from these ten polypeptides, the 2–D patterns for epiblast and ectoderm were practically identical, and distinguishable from those of other tissues by a lack of any unique polypeptides. On the other hand, stage-4 endoblast and stage-12 endoderm differed in the expression of many polypeptides. One polypeptide was found that may be considered as a marker of mesodermal cell type, as it was present in lateral plate, segmental plate and somitic mesoderm, but not in tissues of the other germ layers. Lateral plate could be distinguished from the other mesodermal tissues in the expression of a number of polypeptides, but the similarity in the 2–D patterns for segmental plate and somites suggest that the separation of somites from the anterior end of the segmental plate is not accompanied by the synthesis of new polypeptides.

Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic mice

1993 ◽  
Vol 13 (6) ◽  
pp. 3176-3190
Author(s):  
C Byrne ◽  
E Fuchs
Keyword(s):  
Transgenic Mice ◽  
Transcription Initiation ◽  
Gene Segment ◽  
Hair Follicles ◽  
Cell Type ◽  
Outer Root Sheath ◽  
Specific Expression ◽  
Root Sheath ◽  
Stratified Epithelia

Keratins K5 and K14 form the extensive intermediate filament network of mitotically active basal cells in all stratified epithelia. We have explored the regulatory mechanisms governing cell-type-specific and differentiation stage-specific expression of the human K5 gene in transiently transfected keratinocytes in vitro and in transgenic mice in vivo. Six thousand base pairs of 5' upstream K5 sequence directed proper basal cell-specific expression in all stratified epithelia. Surprisingly, as few as 90 bp of the K5 promoter still directed expression to stratified epithelia, with expression predominantly in epidermis, hair follicles, and tongue. Despite keratinocyte-preferred expression, the truncated K5 promoter displayed departures from basal to suprabasal expression in epidermis and from outer root sheath to inner root sheath expression in the follicle, with some regional variations in expression as well. To begin to elucidate the molecular controls underlying the keratinocyte specificity of the truncated promoter, we examined protein-DNA interactions within this region. A number of keratinocyte nuclear proteins bind to a K5 gene segment extending from -90 to +32 bp and are functionally involved in transcriptional regulation in vitro. Interestingly, several of these factors are common to both the K5 and K14 promoters, although they appear to be distinct from those previously implicated in keratinocyte specificity. Mutagenesis studies indicate that factors binding in the vicinity of the TATA box and transcription initiation are responsible for the cell type specificity of the truncated K5 promoter.

scholarly journals Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate

1981 ◽  
Vol 199 (2) ◽  
pp. 323-333 ◽  
Author(s):  
D G Williams ◽  
M K Johnson
Keyword(s):  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
The Other ◽  
Particulate Fraction ◽  
Molecular Weights ◽  
The Brain ◽  
Neurotoxic Esterase

The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1.

Analysis of proteins synthesizedin vitroby the erythrocytic stages ofPlasmodium knowlesi

Parasitology ◽  
1980 ◽  
Vol 81 (1) ◽  
pp. 177-198 ◽  
Author(s):  
A. A. McColm ◽  
P. G. Shakespeare ◽  
P. I. Trigg
Keyword(s):  
Developmental Stages ◽  
Plasmodium Knowlesi ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electrophoretic Analysis ◽  
The Other ◽  
Blue Staining ◽  
Coomassie Blue Staining

SUMMARYStudies were performed to identify specific parasite proteins synthesized withinPlasmodium knowlesi-infected rhesus erythrocytes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of whole parasites freed from the host erythrocyte by immune lysis, of membranous and cytoplasmic parasite fractions, and of isolated merozoites, detected several parasite-specific components after Coomassie Blue staining of the separated proteins. However, significant contamination with host erythrocyte material generally occurred, particularly in the whole parasite and parasite membrane preparations. Improved identification of plasmodial proteins was subsequently afforded by a radioisotope labelling technique in which parasitized erythrocytes were cultivatedin vitrowith [3H] isoleucine prior to electrophoretic analysis. Of 11 principal labelled peaks ranging in molecular weight from approximately 17000 to 145000 which were detected upon electrophoresis of whole parasites harvested from culture, all were observed in the cytoplasmic fraction while at least 5 were also associated with the membranous cell fraction. Analysis of different developmental stages of the intra-erythrocytic parasite revealed no significant stage-specific qualitative variations in the electrophoretic profiles. Quantitatively, however, the middle to late trophozoites incorporated more [3H] isoleucine into protein than the other intra-erythrocytic stages. Analysis of merozoites purified from labelled schizonts showed a protein pattern similar to the other stages. This confirmed that host components did not contribute to the labelling pattern and that none of the labelled proteins were specific to the residual cytoplasm remaining after merozoite formation.

The origin and movement of nephrogenic cells in the chick embryo as determined by radioautographic mapping

Development ◽  
1970 ◽  
Vol 24 (2) ◽  
pp. 367-380
Author(s):  
Glenn C. Rosenquist
Keyword(s):  
Chick Embryo ◽  
Tritiated Thymidine ◽  
Anterior Part ◽  
The Other ◽  
Lateral Plate ◽  
Intermediate Mesoderm ◽  
Somite Stage ◽  
Stage Of Development ◽  
Stage Embryo ◽  
Area Pellucida

The origin of the presumptive nephrogenic cells in the epiblast of the chick embryo was traced by radioautographic analysis of the movements of tritiated thymidine-labelled grafts excised from medium-streak to 5-somite stage embryos and transplanted to epiblast, streak, and the endoderm-mesoderm layer of similarly staged recipient embryos. The nephrogenic cells originate near the area pellucida margin of the medium-streak-stage embryo, migrate toward the streak, and are invaginated about one-third to one-half the distance from the anterior to the posterior end of the streak, between the definitive-streak and I - to 4-somite stages. Their route into mesoderm is along a relatively narrow pathway between the cells migrating to the paraxial or presomite mesoderm on one side, and those destined for the proximal limbs of the lateral plate on the other. The cells which will form the anterior part of the intermediate mesoderm are the most medially placed cells in epiblast, reach the streak at an earlier stage of development, and are the first nephrogenic cells to migrate into mesoderm. After about the 17– to 19-somite stage, cells from this group which have formed the pronephric cord or duct begin to move posteriorly in relation to the rest of the intermediate mesoderm, toward the future cloaca. The last nephrogenic cells to leave epiblast and enter the streak and mesoderm are those destined for the posterior end of the intermediate mesoderm. This group of cells surrounds the posteriorly migrating pronephric (Wolffian) duct and differentiates into mesonephros.

Synthesis of globin chains in the erythropoietic sites of the early chick embryo

Development ◽  
1983 ◽  
Vol 77 (1) ◽  
pp. 153-165
Author(s):  
L. Fucci ◽  
C. Cirotto ◽  
L. Tomei ◽  
G. Geraci
Keyword(s):  
Chick Embryo ◽  
Relative Concentration ◽  
The Other ◽  
Specific Mechanism ◽  
In Ovo ◽  
Globin Chains ◽  
Immunofluorescence Labelling ◽  
Α Chain

The synthesis of globins in the chick embryo before the onset of circulation has been studied in situ by specific immunofluorescence labelling of embryonic sections and by labelling newly synthesized proteins in ovo and in vitro in embryonic explants with [3H]leucine. The presence of major primitive haemoglobins is observed by 28 h of incubation. The minor primitive haemoglobins become detectable by immunofluorescence after 40 h of development, shortly before the onset of circulation. 3H-labelling shows that one definitive α chain is synthesized, though in low concentration, from the initial globin detection. The other definitive α chain is observed in embryos of at least 40 h of development. The relative concentration of the two definitive α chains changes rapidly with development indicating a specific mechanism of regulation. An erythropoietic site is observed in the wall of the dorsal aorta in embryos of about 45–50 h of development. From the initial detection, those cells contain all four primitive embryonic haemoglobins, in contrast to what is observed for the cells of the blood islands.

Cell Type- and Isotype-Specific Expression and Regulation of β-Tubulins in Primary Olfactory Ensheathing Cells and Schwann Cells In Vitro

2013 ◽  
Vol 38 (5) ◽  
pp. 981-988 ◽  
Author(s):  
Mohamed Omar ◽  
Florian Hansmann ◽  
Robert Kreutzer ◽  
Mihaela Kreutzer ◽  
Gudrun Brandes ◽  
...  
Keyword(s):  
Schwann Cells ◽  
Olfactory Ensheathing Cells ◽  
Cell Type ◽  
Specific Expression ◽  
Ensheathing Cells

HETEROKARYON ANALYSIS OF THE GENETIC CONTROL OF PIGMENT SYNTHESIS IN CHICK EMBRYO MELANOCYTES

Genetics ◽  
1982 ◽  
Vol 102 (3) ◽  
pp. 557-569
Author(s):  
L M Wilkins ◽  
J A Brumbaugh ◽  
J W Moore
Keyword(s):  
Chick Embryo ◽  
Genetic Control ◽  
Regulatory Elements ◽  
The Other ◽  
Wild Type ◽  
Mutant Type ◽  
Pigment Synthesis ◽  
Dominant Mutant

ABSTRACT The genetic control of pigmentation was analyzed using five unlinked mutants, namely, c, pk, Bl, ey and I. Each mutant blocks or reduces pigmentation. Chick melanocyte cultures of each mutant type were fused to produce all ten possible pair combinations of nondividing heterokaryons. Heterokaryons were identified autoradiographically. (One partner in each pair was labeled with 3H-thymidine.) Crosses produced comparable pairs of double heterozygotes that were analyzed in vivo and in vitro. Heterokaryon pairs were compared to their corresponding double heterozygotes.—Some combinations showed complementation and produced wild-type pigment. Others showed noncomplementation having little or no pigment. Double heterozygotes complemented each other except in the cases involving the dominant mutant, I. Four heterokaryon pairs gave different results from their corresponding double heterozygotes. The pk-Bl and pk-ey combinations failed to complement as heterokaryons but did complement as double heterozygotes. On the other hand, the I-c and I-Bl combinations complemented as heterokaryons but not as double heterozygotes. Based on these differences it is hypothesized that the pk and I loci are nuclearly restricted regulatory elements. Examples in the literature from other systems are cited to support such a hypothesis.

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.

scholarly journals Changes in the surface membrane during myoblast fusion

1980 ◽  
Vol 190 (3) ◽  
pp. 647-652 ◽  
Author(s):  
D H Curtis ◽  
K J Micklem ◽  
K Gill ◽  
C A Pasternak
Keyword(s):  
Amino Acids ◽  
Chick Embryo ◽  
Surface Membrane ◽  
Surface Protein ◽  
Myoblast Fusion ◽  
The Other ◽  
Methyl Glucoside ◽  
Simple Diffusion ◽  
Approximate Doubling

1. During fusion of chick-embryo myoblasts in culture, the surface membrane is affected as follows. Uptake of 2-aminoisobutyrate and 2-deoxyglucose, each of which is concentrated 20-fold relative to its concentration in the medium, is unaltered; uptake of alpha-methyl glucoside and choline (15 mM), each of which equilibrates relative to its concentration in the medium, approximately doubles. An approximate doubling also occurs in iodinatable surface protein (and in total protein) and in cell surface area as judged by light-microscopy. Adenylate cyclase (in the absence or the presence of fluoride) increases by more than 2-fold. 2. It is concluded that, during myoblast fusion cells increase in size, and this is reflected in an increased rate of simple diffusion; the rate of facilitated processes such as the uptake of amino acids and sugars, on the other hand, remains unaltered, though the activity of certain enzymes is increased. These results indicate that specific changes in the function of surface membrane occur during myoblast fusion in vitro.

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