Two-dimensional Polyacrylamide Gel Analysis Research Articles

A Proteomics Approach To Quantify Protein Levels Following RNA Interference: Case Study with Glutathione Transferase Superfamily from the Model Metazoan Caenorhabditis elegans

Loss-of-function phenotypic analysis via interference RNA (RNAi) technology is a revolutionary approach to assigning gene function. While transcript-based methodologies commonly validate RNAi gene suppression investigations, protein-based validation is less developed. This report illustrates the potential for two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-DE) and gel analysis to quantify protein levels following RNAi. This case study involves three glutathione transferase (GST) genes targeted by RNAi from the model organism Caenorhabditis elegans.

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3--10 days was approximately 1.08 +/- 0.04 microg/female and 0.1 +/- 0.05 microg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

Monoclonal antibody against a cell wall marker protein for embryogenic potential of Dactylis glomerata L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.

Association of a Major Protein Antigen of Mycoplasma arthritidis with Virulence

Mycoplasma arthritidis causes acute polyarthritis in rats and chronic proliferative arthritis in mice. M. arthritidis-induced arthritis serves as a model for arthritis caused by infectious agents and as a model for examining the role of the superantigen MAM (M. arthritidis T-cell mitogen) in the development of autoimmunity. M. arthritidis strain 158-1 is a spontaneous mutant of strain 158 that has a drastic reduction in virulence. We show that the mutant is missing a major antigen of 47 kDa (P47) and has acquired a protein of 67 kDa (P67). P47 and P67 partitioned into the detergent phase by extraction with Triton X-114. Coomassie blue staining of sodium dodecyl sulfate-polyacrylamide gels show that P67 is produced in abundance. Analysis of gel-purified P67 by mass spectrometry led to its identification as a lipoprotein (the open reading frame [ORF] 619 gene product) predicted from the genome sequence of M. arthritidis. PCR analysis of genomic DNA from 158 and 158-1 indicates that P47 and P67 are encoded by the same ORF 619 gene and differ only in the number of repeats in a tandem repeat region. By two-dimensional polyacrylamide gel analysis, no protein differences were detectable between 158 and 158-1 other than P47 and P67. Collectively, the data suggest that the tandem repeat region of P47 and P67 influences disease outcome.

Analysis by matrix assisted laser desorption/ ionisation-time of flight mass spectrometry of the post-translational modifications of α1-antitrypsin isoforms separated by two- dimensional polyacrylamide gel electrophoresis

The state of protein glycosylation in terms of occupation of potential N-linked glycosylation sites (macroheterogeneity) and type of glycosylation at that site (microheterogeneity) is important when investigating the consequences of aberrant glycosylation in the pathophysiology of disease. Protocols have been developed to permit characterisation of the site-specific glycosylation of individual isoforms of glycoproteins after separation by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and analysis of the peptide mixture by peptide mass fingerprinting using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF). High resolution of the individual isoforms of α1- antitrypsin was achieved by using narrow range (4.5–5.5) pI strips. The individual isoforms were then subjected to sequential digestion with a recombinant N-glycanase followed by a protease. Using this strategy it was possible not only to increase the coverage of the amino acid sequence but also to monitor the occupancy of all three putative N-linked glycosylation sites. Glycans were enzymatically released from α1-antitrypsin which had been separated in gels formed with a low percentage of bis-acrylamide cross-linker and analysed. Profiles of the N-linked glycans of the individual isoforms of α1-antitrypsin were obtained by MALDI-TOF.

Analysis by matrix assisted laser desorption/ionisation-time of flight mass spectrometry of the post-translational modifications of alpha 1-antitrypsin isoforms separated by two-dimensional polyacrylamide gel electrophoresis.

The state of protein glycosylation in terms of occupation of potential N-linked glycosylation sites (macroheterogeneity) and type of glycosylation at that site (microheterogeneity) is important when investigating the consequences of aberrant glycosylation in the pathophysiology of disease. Protocols have been developed to permit characterisation of the site-specific glycosylation of individual isoforms of glycoproteins after separation by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and analysis of the peptide mixture by peptide mass fingerprinting using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF). High resolution of the individual isoforms of alpha 1-antitrypsin was achieved by using narrow range (4.5-5.5) p/strips. The individual isoforms were then subjected to sequential digestion with a recombinant N-glycanase followed by a protease. Using this strategy it was possible not only to increase the coverage of the amino acid sequence but also to monitor the occupancy of all three putative N-linked glycosylation sites. Glycans were enzymatically released from alpha 1-antitrypsin which had been separated in gels formed with a low percentage of bis-acrylamide cross-linker and analysed. Profiles of the N-linked glycans of the individual isoforms of alpha 1-antitrypsin were obtained by MALDI-TOF.

Identification of acquired thermotolerance deficiency within the ditelosomic series of `Chinese Spring' wheat

The relative contribution of individual heat shock proteins to acquired thermotolerance was evaluated through analysis of chromosomal deletions in a ditelosomic series of the hexaploid wheat ( Triticum aestivum L.) cultivar `Chinese Spring'. This study describes the identification of a line within this ditelosomic series that exhibited a reduced level of acquired thermotolerance. Changes in the temperature sensitivity of chlorophyll accumulation were used as an indicator of acquired thermotolerance. The temperature providing maximum chlorophyll accumulation was 30 °C in leaves under continuous light. A 30-min challenge temperature of 48 °C prior to the light exposure was shown to inhibit subsequent chlorophyll accumulation. Preincubation at 40 °C for 4 h before the 30-min 48 °C challenge triggered the acquired thermotolerance system of the plant resulting in chlorophyll accumulation upon exposure to light. Evaluation of the ditelosomic series revealed reductions in acquired thermotolerance levels in the DT7DS line relative to controls. Two-dimensional SDS polyacrylamide gel analysis was used to identify reduction in the level of two low molecular mass heat shock proteins in DT7DS.

Dominance ofFrankiastrains in stands ofAlnus incanasubsp.rugosaandMyrica pensylvanica

To address issues of dominance and diversity of Frankia spp. strains, we sequenced 16S rRNA genes from root nodules and strains collected from Alnus incana subsp. rugosa (Du Roi) R.T. Clausen and Myrica pensylvanica Loisel. stands. Of 22 strains isolated previously from A. incana, 16 had the same partial rDNA sequence; the remaining 6 strains composed five additional groups. The groups identified by 16S rDNA analysis corresponded to phenotypic groups established previously by one- and two-dimensional polyacrylamide gel analysis, colony and hyphal morphology, and carbon source utilization patterns. Dominance of one strain was also evident in nodules collected from a single M. pensylvanica stand. The dominant strain had a partial 16S rDNA sequence identical to that of Frankia alni strain CpI1.Key words: Frankia, Myrica, Alnus, actinorhizal, root nodules, nitrogen fixation, symbiosis, 16S rRNA.

Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase.

Recent studies have shown that the p70(s6k)/p85(s6k) signaling pathway plays a critical role in cell growth by modulating the translation of a family of mRNAs termed 5'TOPs, which encode components of the protein synthetic apparatus. Here we demonstrate that homozygous disruption of the p70(s6k)/p85(s6k) gene does not affect viability or fertility of mice, but that it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70(s6k)/p85(s6k)-deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum-induced S6 phosphorylation and translational up-regulation of 5'TOP mRNAs were equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70(s6k)/p85(s6k)-deficient and wild-type mice. A search of public databases identified a novel p70(s6k)/p85(s6k) homolog which contains the same regulatory motifs and phosphorylation sites known to control kinase activity. This newly identified gene product, termed S6K2, is ubiquitously expressed and displays both mitogen-dependent and rapamycin-sensitive S6 kinase activity. More striking, in p70(s6k)/p85(s6k)-deficient mice, the S6K2 gene is up-regulated in all tissues examined, especially in thymus, a main target of rapamycin action. The finding of a new S6 kinase gene, which can partly compensate for p70(s6k)/p85(s6k) function, underscores the importance of S6K function in cell growth.

Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin

The cDNA coding for mouse fibroblast tropomyosin isoform 2 (TM2) was placed into a bacterial expression vector to produce a fusion protein containing glutathione-S-transferase (GST) and TM2 (GST/TM2). Glutathione-Sepharose beads bearing GST/TM2 were incubated with [35S]methionine-labeled NIH 3T3 cell extracts and the materials bound to the fusion proteins were analyzed to identify proteins that interact with TM2. A protein of 10 kD was found to bind to GST/TM2, but not to GST. The binding of the 10-kD protein to GST/TM2 was dependent on the presence of Ca2+ and inhibited by molar excess of free TM2 in a competition assay. The 10-kD protein-binding site was mapped to the region spanning residues 39-107 on TM2 by using several COOH-terminal and NH2-terminal truncation mutants of TM2. The 10-kD protein was isolated from an extract of NIH 3T3 cells transformed by v-Ha-ras by affinity chromatography on a GST/TM2 truncation mutant followed by SDS-PAGE and electroelution. Partial amino acid sequence analysis of the purified 10-kD protein, two-dimensional polyacrylamide gel analysis and a binding experiment revealed that the 10-kD protein was identical to a calcium-binding protein derived from mRNA named pEL98 or 18A2 that is homologous to S100 protein. Immunoblot analysis of the distribution of the 10-kD protein in Triton-soluble and -insoluble fractions of NIH 3T3 cells revealed that some of the 10-kD protein was associated with the Triton-insoluble cytoskeletal residue in a Ca(2+)-dependent manner. Furthermore, immunofluorescent staining of NIH 3T3 cells showed that some of the 10-kD protein colocalized with nonmuscle TMs in microfilament bundles. These results suggest that some of the pEL98 protein interacts with microfilament-associated nonmuscle TMs in NIH 3T3 cells.

Signal transduction in Coprinus congregatus: Evidence for the involvement of G proteins in blue light photomorphogenesis

This paper reports the presence of several G proteins and light-sensitive GTP-binding proteins in the fungus Coprinus congregatus, a filamentous eukaryote. (Mono)ADP-ribosylation experiments with crude membranes in the presence of the (poly)ADP-ribosyltransferase inhibitor, 3-amino-benzamide, resulted in the detection of a cholera toxin substrate of 52 kDa and two pertussis toxin substrates, 33 and 39 kDa. Two-dimensional polyacrylamide gel analysis of GTP-binding proteins exposed in vivo to [35S]-labeled guanosine 5'-[gamma-thio]-triphosphate in the presence or absence of light demonstrated light enhanced analog binding. These results support the concept of the involvement of G proteins in phototransduction in C. congregatus.

Mobility of positioned nucleosomes on 5 S rDNA

We report on a dynamic aspect of nucleosome positioning, in the absence of transcription-related events, on sea urchin 5 S rDNA. On tandem repeats of nucleosome length DNA of this strongly positioning sequence, histone octamers assemble in one dominant position surrounded by minor positions, ten base-pairs apart and therefore with identical rotational setting of the DNA coil. The existence of this cluster of positions, determined using micrococcal nuclease is confirmed by the results from DNase I footprinting and restriction enzyme analysis. The results from these techniques and from two-dimensional nucleoprotein polyacrylamide gel analysis indicate that the cluster of octamer positions is in dynamic equilibrium, in low ionic conditions, suggesting that the minor positions reflect fluctuations around the major nucleosome site. Histone octamer mobility appears to be temperature dependent and is reversibly inhibited by Mg2+.

RNA transcription and translation in the hearts of normal and cardiomyopathic Syrian hamsters

The cardiomyopathic Syrian hamster has an autosomal recessive defect that results in the development of an early onset cardiac myopathy leading to cardiac dysfunction and, eventually, complete heart failure. To assess the regulatory mechanisms modulating gene expression in the normal and myopathic myocardium, we investigated both RNA transcription and translation. Our results indicated that the incorporation of [3H]UMP into myocardial cell nuclear RNA decreased 10-fold from 7 to 210 days of age in the normal Syrian hamster. The incorporation of [3H]UMP was approximately 50% lower in the cardiomyopathic as compared with the normal Syrian hamster. RNA translation, as assessed by rabbit reticulocyte lysate in vitro translation, indicated that a coordinated 50% decrease in RNA translation occurred in normal Syrian hamster from 7 to 210 days of age. A further reduction of 20% in translation was found in cardiomyopathic Syrian hamster ventricular RNA translation as compared with matched random bred control groups. Two-dimensional polyacrylamide gel analysis of cell-free translated protein products demonstrated two myocardial peptides that were found to be consistently altered when the normal and cardiomyopathic Syrian hamsters were compared. These results indicate that transcription and translation decrease with age and that these processes are further downregulated, in an additive manner, with the genesis of the disease process.

Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1α reveals a new post-translational protein modification

Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988) J. Biol. Chem. 263, 8244-8252). In particular, the protein was enriched in cytosolic and microsomal fractions relative to plasma membrane and thus did not appear to correspond to the class of proteins with glycoinositol phospholipid anchors, the only post-translational protein modification involving ethanolamine that had been described previously. Two-dimensional polyacrylamide gel analysis involving isoelectric focusing followed by electrophoresis in sodium dodecyl sulfate indicated that the protein was very basic, and nitrocellulose blots of one- and two-dimensional gels subjected to 3H autoradiography and immunostaining with antisera to purified rabbit elongation factor (EF) 1 alpha revealed that the protein was EF-1 alpha. Copurification of rabbit EF-1 alpha and the [3H]ethanolamine-labeled protein from K562 cells further supported this identification. Analysis of tryptic fragments produced from the copurified proteins by reverse-phase high pressure liquid chromatography showed two radiolabeled peptides. Amino acid analysis demonstrated 1 residue of ethanolamine in each peptide, and peptide sequencing revealed that the ethanolamine-containing component(s) was attached to Glu301 and Glu374 in the EF-1 alpha protein sequence deduced from a human EF-1 alpha cDNA. These data confirm a new class of post-translational protein modifications involving ethanolamine.

Water Deficit-Induced Changes in Abscisic Acid, Growth, Polysomes, and Translatable RNA in Soybean Hypocotyls

Soybean seedlings (Glycine max L.) were germinated and dark-grown in water-saturated vermiculite (water potential = -0.01 megapascal) for 48 hours, then transferred either to water-saturated vermiculite or to low water potential vermiculite (water potential = -0.30 megapascal). A decrease in growth rate was detectable within 0.8 hour post-transfer to low water potential vermiculite. A fourfold increase in the abscisic acid content of the elongating region was observed within 0.5 hour. At 24 hours post-transfer, hypocotyl elongation was severely arrested and abscisic acid reached its highest measured level: 3.7 nanograms per milligram dry weight (74-fold increase). A comparison of the polyA(+) RNA populations isolated at 24 hours post-transfer from the elongating region of water-saturated and low water potential vermiculite-grown seedlings was made by two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gel analysis of in vitro translation products. It revealed both increases and decreases in the relative amounts of a number of translation products. Rewatering seedlings grown in low water potential vermiculite at 24 hours post-transfer led to a total recovery in growth rate within 0.5 hour, while abscisic acid in the elongating hypocotyl region required 1 to 2 hours to return to uninduced levels. Application of 1.0 millimolar (+/-) abscisic acid to well-watered seedlings resulted in a 48% reduction in hypocotyl growth rate during the first 2 hours after treatment. Plants treated with abscisic acid for 24 hours had a lower polysome content than control plants. However, hypocotyl growth inhibition in abscisic acid-treated seedlings preceded the decline in polysome content.

A nuclear protein associated with cell divisions in plants

A nuclear protein, present in carrot meristems and rapidly proliferating cultured cells of carrot (Daucus carota L.) has been identified by the use of a monoclonal antibody (MAb 21D7). By combining the techniques of two-dimensional polyacrylamide gel analysis and blotting separated proteins onto nitrocellulose sheets, it was shown that the antibody detected a single polypeptide of apparent molecular mass (M r) of 45000 and an isoelectric focusing point (pI) of 6.7. This protein was found by subcellular fractionation and immunofluorescence to be highly concentrated in the nucleoli of somatic and zygotic embryos of a wide range of plants. It was not detectable in logarthmically growing cells ofEscherichia coli, yeast, embryos ofDrosophila melanogaster or cultured C3H mouse cells. These data indicate that this protein is a highly conserved non-histone protein associated with nuclei of rapidly dividing plant cells.

Plant Plasma Membrane Proteins

A major 75 kD protein group from the tomato plasma membrane was semipurified on polyacrylamide gels and used to raise a rabbit antiserum. The resulting antiserum recognized a single 75 kilodalton band from phase partitioned tomato plasma membrane (from both suspension cells and mature, green fruit) after resolution on one-dimensional polyacrylamide gels. Two-dimensional polyacrylamide gel analysis of proteins from tomato plasma membrane showed that the 75 kilodalton antiserum recognized a group of proteins ranging from 63.1 to 88.2 kilodaltons (mean = 75.6 kilodaltons) and with isoelectric point values ranging from 5.7 to 6.3. No other spots were visible on the two-dimensional blots. This antiserum was shown to bind protoplast surface epitopes by indirect immunofluorescence. The presence of this protein group in both monocotyledonous and dicotyledonous plants was established by immunoblotting the tomato 75 kilodalton antiserum against proteins obtained from plasma membrane-enriched fractions from corn roots and soybean roots. The data suggest that this 75 kilodalton protein group is a major proteinaceous component of the plant plasma membrane.

Transcript levels and translational control of hsp70 synthesis in Xenopus oocytes.

Until recently it was believed that Xenopus oocytes respond to heat shock by synthesizing the 70-kD heat shock protein hsp70 and that, uniquely amongst animal cell types, this response is mediated entirely at the translational level. This view has now been challenged and we present data that reevaluate the involvement of translational control in the heat shock response of Xenopus oocytes. RNase mapping shows that up to 13 pg of hsp70A and hsp70B mRNA are accumulated by fully grown oocytes in the absence of heat shock. These transcripts are retained stably during maturation, fertilization, early cleavage, and following heat shock. However, no hsp70 protein synthesis can be detected by two-dimensional polyacrylamide gel analysis of [35S]methionine-labeled proteins from completely defolliculated oocytes, either before or during heat shock. Oocytes injected with hsp70A DNA rapidly accumulate high levels of hsp70 mRNA in their cytoplasm at normal temperature. During heat shock these oocytes accumulate more transcripts, but they remain in the nucleus and cytoplasmic levels remain constant. Translation of hsp70 from these transcripts is readily detectable at non-heat shock and heat shock temperatures. We conclude that (1) "exogenous" hsp70 transcripts are efficiently translated and not masked at normal temperatures in oocytes, and (2) oocytes are able to selectively translate hsp70 mRNA during heat shock.

Altered type I protein kinase in adhesion defective CHO cell variants.

ADv cells are Chinese hamster ovary (CHO) cell variants which cannot adhere to fibronectin coated substrata (Harper & Juliano: J. Cell Biol., 1980; Nature 1981a,b). We have shown that the defect in some clones of ADv cells is distal to the initial interaction between fibronectin and its cell surface receptors (Cheung and Juliano: Exp. Cell Res., 1984), and that it extends to fibronectin mediated aggregation and endocytosis. The adhesion defect in some ADv clones can be corrected by raising intracellular cAMP levels (Cheung & Juliano: J. Cell Physiol., 1985). Here we examine the protein kinase activities and phosphorylation patterns in an adhesion defective variant clone ADv F11CA11. Analysis of the cAMP dependent protein kinase activity (cAdPK) in crude extracts of F11CA11 cells shows an apparent increase in K (activation) as compared to wild type (WT) CHO cell extracts. Further, the DE-52 cellulose chromatography profile of cAdPK in the F11CA11 variant is markedly different from WT in that the type I cAdPK peak elicited by 1 microM cAMP is essentially missing in F11CA11, while the type II cAdPK peak is similar to that in WT. Raising the cAMP level to 100 microM elicits a type I peak in F11CA11 with about 45% of the activity of the WT peak. Binding studies with 3H-cAMP reveal that the type I peak in F11CA11 has a Kd of 1.7 X 10(-8) M as compared to 2.0 X 10(-9) M for WT, whereas the type II peak Kd is approximately 1 X 10(-9) M for both WT and F11CA11. Two-dimensional polyacrylamide gel analysis of 32Pi labeled WT cells and F11CA11 cells with or without cAMP treatment reveals the presence of a protein(s) of 50 kilodaltons which is phosphorylated in WT cells and in cAMP treated F11CA11 cells but not in untreated F11CA11 cells. These findings, coupled with our previous observations, strongly indicate that the adhesion defect in ADvF11CA11 cells is associated with an altered type I cAdPK having lower affinity for cAMP. At normal cellular cAMP levels this enzyme fails to phosphorylate one or more critical protein substrates; however, by raising internal cAMP levels, the defect can be overcome. Thus type I cAdPK seems to play an important role in the regulation of fibronectin mediated cell adhesion, cell aggregation, and endocytosis.

Differentiation of embryonic chick feather-forming and scale-forming tissues in transfilter cultures

The dermal-epidermal tissue interaction in the chick embryo, leading to the formation of feathers and scales, provides a good experimental system to study the transfer between tissues of signals which specify cell type. At certain times in development, the dermis controls whether the epidermis forms feathers or scales, each of which are characterized by the synthesis of specific β-keratins. In our culture system, a dermal effect on epidermal differentiation can still be observed, even when the tissues are separated by a Nuclepore filter, although development is abnormal. Epidermal morphological and histological differentiation in transfilter cultures are distinct and recognizable, more closely resembling feather or scale development, depending on the regional origin of the dermis. Differentiation is more advanced when epidermis is cultured transfilter from scale dermis than from feather dermis, as assessed by morphology and histology, as well as the expression of the tissue-specific gene products, the β-keratins. Two-dimensional polyacrylamide gel analysis of the β-keratins reveals that scale dermis cultured transfilter from either presumptive scale or feather epidermis induces the production of 7 of the 9 scale-specific β-keratins that we have identified. Feather dermis, although less effective in activating the feather gene program when cultured transfilter from either presumptive feather or scale epidermis, is able to turn on the synthesis of 3 to 6 of the 18 feather-specific β-keratins that we have identified. However, scale epidermis in transfilter recombinants with feather dermis also continues to synthesize many of the scale-specific β-keratins. Using transmission and scanning electron microscopy, we detect no cell contact between tissues separated by a 0.2-μm pore diameter Nuclepore filter, while 0.4-μm filters readily permit cell processes to traverse the filter. We find that epidermal differentiation is the same with either pore size filter. Furthermore, we do not detect a basement membrane in transfilter cultures, implying that neither direct cell contact between dermis and epidermis, nor a basement membrane between the tissues is required for the extent of epidermal differentiation that we observe.