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The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl− channels and Cl−/H+ exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3−/− mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.

Development of cell lines for biotherapeutic protein production requires screening large numbers of clones to identify and isolate high producing ones. As such, stable cell line generation is a time- and resource-intensive process. There is an increasing need to enhance the selection efficiency of high-yielding clonal cell lines for cell line development projects by using high throughput screening of live cells for markers predictive of productivity. Single cell deposition by fluorescence activated cell sorting (FACS) is a commonly performed method for cloning to generate cell lines derived from a single recombinant cell. We have developed a novel strategy to identify higher productivity cells at the FACS step by leveraging a simple viable cell staining method that detects mitochondrial membrane potential (Ψm), a key indicator of cellular metabolic activity. We chose a dual-emission dye (Mito-ID, Enzo Life Sciences) that fluoresces green and orange in living cells with the intensity of the orange fluorescence being dependent on the cells Ψm status. Using available clonal cell lines with known productivity, or stable transfectant pools, we evaluated Ψm of cell populations with Mito-ID dye. We determined that the intensity of the Ψm fluorescent signal correlates with the known fed-batch titers of the producer clones, and that cell sorting based on an optimal Ψm staining intensity selectively enriches for higher producing clones from nonclonal transfectant pools. These clones are phenotypically stable for recombinant protein production. Furthermore, the strategy has been successfully applied to identifying higher producing cell lines for a range of antibody molecular formats. Using this method, we can combine an enriching step with the cloning step for high producers, thereby saving time and resources in cell line development.

Atypical presentations of complex multisystem disorders may elude diagnosis based on clinical findings only. Appropriate diagnostic tests may not be available or available tests may not provide appropriate coverage of relevant genomic regions for patients with complex phenotypes. Clinical whole-exome/-genome sequencing is often considered for complex patients lacking a definitive diagnosis.A boy who is now 7 years old presented as a newborn with congenital ichthyosis. At 6 weeks of age, he presented with failure to thrive and hypoparathyroidism. At 4 years of age, he was diagnosed with sensorineural hearing loss. Whole-genome sequencing identified novel mutations in GATA3, which causes HDR syndrome (hypoparathyroidism and deafness), and STS, which causes X -linked congenital ichthyosis.Whole-genome sequencing led to a definitive clinical diagnosis in a case where no other clinical test was available for GATA3, and no sequencing panel would have included both genes because they have disparate phenotypes. This case demonstrates the power of whole-genome (or exome) sequencing for patients with complex clinical presentations involving endocrine abnormalities.

The manufacturing process for biotherapeutics is closely regulated by the Food and Drug Administration (FDA), European Medicines Agency (EMA) and other regulatory agencies worldwide. To ensure consistency of the product of a manufacturing cell line, International Committee on Harmonization guidelines (Q5D, 1997) state that the cell substrate should be derived from a single cell progenitor, i.e., clonal.Cell lines in suspension culture may naturally revert to cell adhesion in the form of doublets, triplets and higher order structures of clustered cells. We can show evidence of a single colony from limiting dilution cloning or in semi-solid media, but we cannot determine the number of cells from which the colony originated. To address this, we have used the ViCELL® XR (Beckman Coulter, High Wycombe, UK) cell viability analyzer to determine the proportion of clusters of two or more cells in a sample of the cell suspension immediately prior to cloning. Here, we show data to define the accuracy of the ViCELL for characterizing a cell suspension and summarize the statistical model combining two or more rounds of cloning to derive the probability of clonality. The resulting statistical model is applied to cloning in semi-solid medium, but could equally be applied to a limiting dilution cloning process. We also describe approaches to reduce cell clusters to generate a cell line with a high probability of clonality from a CHO host lineage. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:593-601, 2018.

Chromosomal microarray (CMA) testing provides the highest diagnostic yield for clinical testing of patients with developmental delay (DD), intellectual disability (ID), multiple congenital anomalies (MCA), and autism spectrum disorders (ASD). Despite improved diagnostic yield and studies to support cost-effectiveness, concerns regarding the cost and reimbursement for CMA have been raised because it is perceived that CMA results do not influence medical management.We conducted a retrospective chart review of CMA testing performed during a 12-month period on patients with DD/ID, ASD, and congenital anomalies to determine the proportion of cases where abnormal CMA results impacted recommendations for clinical action.Among 1792 patients, 13.1% had clinically relevant results, either abnormal (n = 131; 7.3%) or variants of possible significance (VPS; n = 104; 5.8%). Abnormal variants generated a higher rate of recommendation for clinical action (54%) compared with VPS (34%; Fisher exact test, P = 0.01). CMA results influenced medical care by precipitating medical referrals, diagnostic imaging, or specific laboratory testing.For all test indications, CMA results influenced medical management in a majority of patients with abnormal variants and a substantial proportion of those with VPS. These results support the use of CMA as a clinical diagnostic test that influences medical management for this patient population.

Background Variants in the desmin gene (DES) are associated with desminopathy; a myofibrillar myopathy mainly characterized by muscle weakness, conduction block, and dilated cardiomyopathy. To date, only ~50 disease-associated variants have been described, and the majority of these lead to dominant-negative effects. However, the complete genotypic spectrum of desminopathy is not well established. Case presentation Next-generation sequencing was performed on 51 cardiac disease genes in a proband with profound skeletal myopathy, dilated cardiomyopathy, and respiratory dysfunction. Our analyses revealed compound heterozygous DES variants, both of which are predicted to lead to a loss-of-function. Consistent with recessive inheritance, each variant was identified in an unaffected parent. Conclusions This case report serves to broaden the variant spectrum of desminopathies and provides insight into the molecular mechanisms of desminopathy, supporting distinct dominant-negative and loss-of-function etiologies.

The development of high-efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipid-based transfection reagents has accelerated biology research in studies of gene expression, control of cell growth, and cell lineage. In this unit, DNA transfection is described for adherent mammalian cells (both cells and primary cultures) along with an alternate procedure for enhanced transfection. A protocol is also described for transfection of suspension cells (lymphoid, myeloid and leukemic-derived cells). In addition, transfection of RNA into adherent mammalian cells and DNA transfection into insect cells are presented. Importantly, a detailed procedure is described for optimization of reagents and transfection conditions.

BACKGROUND Pure partial trisomy of chromosome 5q is rare and cases have ranged over the entire region, making it difficult to describe a good phenotypic correlation to the cytogenetic duplication. CASE We present a 4.5-year-old girl with a de novo direct duplication of chromosome 5q15-q23.2. She has moderate developmental delay with lack of speech, microcephaly, and subtle dysmorphic features including prominent forehead, bulbous nose, epicanthic folds, protruding tongue, and slightly posteriorly-rotated ears. CONCLUSIONS A comparison is made with other similar duplication cases reported in the literature and a general description of a proximal 5q duplication phenotype is given, with lack of speech as the principal feature. Birth Defects Research (Part A), 2006. © 2006 Wiley-Liss, Inc.

Female carriers of Duchenne muscular dystrophy (DMD) may demonstrate elevated serum creatine kinase (CK) and reduction of muscle dystrophin in all muscle types. We hypothesized that decreased dystrophin in uterine or pelvic girdle musculature might affect the obstetrical performance of females heterozygous for a dystrophin mutation. We reviewed the outcome of 34 deliveries resulting in 35 children from 13 women who were mothers of males attending a muscular dystrophy clinic. Obstetrical performance was examined retrospectively by chart review and patient contact. Of 35 children, 6 (17%) were delivered in the breech position, which is a fivefold increase above the national standards for term pregnancies. Of the six infants with breech presentation, two were males affected with DMD, one was a female heterozygote, one was a male who died perinatally, and the carrier status of the other two females is unknown. Most DMD affected males (12/14) were delivered in the vertex position. Thus, it is likely that maternal, rather than fetal, muscle weakness was the significant factor in determination of fetal position at term. We speculate that subtle changes in uterine or pelvic girdle muscle tone may contribute to a higher rate of fetal breech position in carriers of the DMD gene.

The development of high-efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. This unit describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using cationic lipid reagents. Curr. Protoc. Mol. Biol. 81:9.4.1-9.4.17. © 2008 by John Wiley & Sons, Inc. Keywords: transfection; luciferase; cationic lipid; cell culture; transgene; transient gene expression; stable gene expression

Skin calcium-binding protein (SCaBP) is a calcium binding protein purified from whole rat skin. It has a molecular weight of approximately 12,000 daltons but migrates at Mr 13,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gels. On nitrocellulose blots of SDS-polyacrylamide gels, 6 different antisera to SCaBP reacted equally well with SCaBP and parvalbumin (PV), an 11,500-dalton calcium-binding protein purified from rat skeletal muscle, which also migrates at Mr 13,000 on SDS-polyacrylamide gels. Rabbit antiserum to muscle PV also recognized both PV and SCaBP, and either protein absorbed specific antibodies against either antigen from both types of antisera. Soluble protein extracts from whole adult rat and mouse skin contained a Mr 13,000 protein which was recognized on nitrocellulose blots of SDS gels by both antisera. Blots of extracts from epidermis, dermis, whole skin, and skin scraped on the dermal side to remove hypodermal tissue revealed that the Mr 13,000 PV/SCaBP cross-reacting antigen was restricted to the hypodermal tissue removed by scraping. Immunofluorescent staining of Bouin-fixed skin sections with these antisera confirmed the localization of PV/SCaBP to the panniculus carnosus, a hypodermal muscle layer. Newborn mouse skin does not contain this antigen. Additional polypeptides of Mr 10,500 and 12,000 on SDS gels of extracts from the epidermis of newborn and adult rats and mice were found to be immunoreactive with anti-SCaBp serum. These polypeptides were not recognized by the PV antiserum, and the reactivity of anti-SCaBP for these antigens was not absorbed by purified PV or SCaBP. Our results indicate that SCaBP is antigenically indistinguishable from PV and is localized in the adult rodent panniculus carnosus, and that antisera to SCaBP are poly-specific, recognizing epidermal proteins in addition to SCaBP/PV.

A gamma gt 11 cDNA expression library representing mouse epidermis mRNA was screened with polyclonal rabbit antiserum directed against 10-13 kDa epidermal antigens that had previously been shown to be regulated during epidermal differentiation. A cDNA clone was detected and isolated and its identity as the coding sequence for one of the antigens was confirmed by translation of hybrid-selected mRNA from mouse epidermis. The cDNA sequence predicted a peptide homologous to the reported sequence of rat epidermis cystatin A, a thiol proteinase inhibitor. This identification was confirmed by cross-reactivity of the gamma clone fusion protein with authentic antiserum to rat epidermis cystatin A. Southern gel analysis showed that mouse DNA may contain several closely related genes homologous to the cystatin probe. An mRNA of about 0.6 kb from epidermis and cultured mouse epidermal cells hybridized with the cystatin probe on northern analysis. The abundance of the message was high in cultured basal cells and was selectively diminished by inducing terminal differentiation in culture with an elevated Ca2+ concentration in the medium. Cystatin message was abundant in chemically induced mouse skin papillomas but reduced in carcinomas. In epidermis, mRNA was localized to the less differentiated basal and lower spinous layers by in situ hybridization. Regulation of expression of cystatin A in epidermis and tumors suggests that it may be important in the control of normal keratinocyte proliferation and differentiation and in malignant conversion.

In order to determine whether pemphigold antigen is synthesized by epidermal cells, and whether other, normal keratinocyte, antigens are synthesized in culture, primary cultures and subsequent subcultures of human epidermal cells derived from neonatal foreskins were studied for the presence of pemphigoid antigen, pemphigus antigen and keratin filaments using indirect immunofluorescence. Twenty-four hr after plating primary cultures, pemphigoid antigen was detected as an asymmetric fluorescence on the cell membranes of rounded cells or a faint fluorescence of the cytoplasm of cells which had spread on the substrate. At later times in primary cultures and in subcultures pemphigoid antigen showed a pattern of coarsely granular fluorescence within or under the cytoplasm of many of the small cells at both the base and expanding periphery of epidermal cell colonies. Pemphigus antigen was detected mainly on the cell membranes of the larger flat cells located more superficially in the colonies. Keratin filaments were detected in cells at all levels of the epidermal colonies. Rounded cells, which had not yet spread on the substrate, displayed bright perinuclear fluorescence with keratin antibody. In cells spread on the substrate, keratin filaments often appeared to line up end-to-end with filaments of neighboring cells. No qualitative changes in fluorescence of any of these antigens could be detected through 6 subcultures. Human fibroblasts in culture did not display any of these three antigens by immunofluorescence. The finding that cultured human epidermal cells displayed these antigens with the same pattern and intensity through 6 passages indicates that pemphigoid antigen, as well as pemphigus antigen and keratin, were newly synthesized in culture.

Complementary DNA (cDNA) clones constructed to the 55, 59 and 67 kilodalton (K) keratins, the major keratins synthesized in newborn mouse epidermis, were used as molecular hybridization probes to examine the expression of these genes in newborn epidermis and normal and malignantly transformed epidermal cells in culture. Transcripts of these three keratin genes are abundant in newborn epidermis. However, primary cultures of epidermal cells contain very low levels of these RNAs. The decreased expression of these keratin genes in primary cells appears to be due to factors within the culture system. Unlike primary-cell cultures, the malignantly transformed cell line Pam 212 synthesizes keratin proteins and mRNAs similar to newborn epidermis, including the 67 K keratin. However, synthesis of the 67 K keratin in Pam 212 cells is modulated by culture factors. Keratin gene expression in another Pam line, 321, differs from that of Pam 212 cells in that decreased expression of these three keratin genes occurs. These results indicate that keratin genes that are normally expressed in vivo in epidermis may be expressed in malignant epidermal cells under conditions that do not permit expression of these genes in nonmalignant primary epidermal cells.

In this study we show that both cultured normal human epidermal cells (EC) and a human squamous cell carcinoma (SCC) cell line produce a thymocyte-activating factor (ETAF). EC-ETAF and SCC-ETAF both have a Mr of 15,000 and were eluted from chromatofocusing at the same isoelectric points of 7.2, 5.8, and 5.0. Both activities were maintained at alkaline pH and were destroyed at temperatures above 60 degrees C. In addition to stimulating thymocyte proliferation, human ETAF exhibited a variety of other pertinent biologic activities. Although EC-ETAF or SCC-ETAF by themselves exhibited no T-cell growth factor activity, both ETAF preparations enhanced Interleukin 2 production by cultured human peripheral blood lymphocytes when stimulated with polyclonal T-cells stimulants (Concanavalin A and phorbol myristate acetate). Human ETAF also was chemotactic for rabbit polymorphonuclear leukocytes and was directly mitogenic for cultured human dermal fibroblasts. Injection of human ETAF into C3H/HeJ mice, resulted in inducing serum amyloid A (SAA) production by murine hepatocytes. The thymocyte growth-enhancing activity, the fibroblast-stimulating activity, and the SAA-inducing capacity of ETAF all coeluted off AcA54 gel. These biologic as well as biochemical properties of human keratinocyte-derived ETAF are identical with those of human macrophage-derived Interleukin 1. The ability of keratinocytes to release an immunomodulating factor with such diverse consequences may play an important role in normal wound healing and in diseases involving epithelial tissues.

Proteins from mouse epidermis cytosol extracts react on immunoblots with a polyclonal rabbit antiserum raised against rat skin calcium-binding protein (SCaBP), a parvalbumin of the panniculus carnosus. Three mouse epidermal proteins with molecular weights between 10–12K, which are distinct from SCaBP, are recognized by the antiserum. The synthesis of these proteins in keratinocyte culture is modulated by Ca ++ , as is the differentiation of the keratinocytes. Proliferating mouse keratinocytes in medium containing 0.07 mm Ca ++ (low Ca ++ ) undergo terminal differentiation when the Ca ++ concentration is elevated to 1.8 mm (high Ca ++ ). Synthesis of the 3 antigens can be demonstrated when soluble extracts of keratinocytes labeled with [ 35 S]methionine in low Ca ++ medium are immunoprecipitated with anti-SCaBP serum. These antigens are not synthesized in cultures of dermal fibroblasts. When keratinocytes are switched to high Ca ++ medium, synthesis of these antigens is greatly diminished over the course of 48–72 h. However, the antigens persist in differentiating cells. When proliferating keratinocytes in low Ca ++ medium are exposed to the tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA), differentiation is induced in a subpopulation of cells, and specific antigen synthesis is transiently inhibited. The inhibition correlates with the time when many cells are differentiating in response to TPA. When proliferating keratinocytes are pulse-labeled with 32 PO 4 , the 11K antigen is phosphorylated and the phosphorylation is not enhanced by TPA exposure. All 3 antigens are synthesized in a reticulocyte lysate preparation with added newborn mouse epidermis messenger RNA or mRNA from keratinocytes cultured in low Ca ++ medium. Thus, these antigens are likely to represent unique proteins rather than processed or degraded ones. The coordinately regulated expression of these antigens associated with the differentiation state of the keratinocytes suggests that these proteins are important in keratinocyte proliferation and differentiation.

Extract: Chloramphenicol (CAP), chloramphenicol succinate (CAP-S), tetracycline (TET), and erythromycin (EM) inhibited synthesis of a mitochondrial enzyme, cytochrome oxidase, in cultured human fibroblasts; the 50% inhibitory concentrations were 7.5, 13, 18, and 350 $mUM respectively, or 2.4, 4.1, 8, and 250 $mUg/ml. In fibroblasts grown in CAP-S, cytochrome oxidase declined exponentially with a half-life of approximately 2.0 days. Oxygen uptake, on the other hand, was unchanged for the first 24–48 hr of exposure to CAP-S, and then declined, but at a much slower rate than did cytochrome oxidase; after 7 days, cytochrome oxidase activity was 6% of the control value, whereas oxygen uptake was 50% of control. From the kinetic data we conclude that cytochrome oxidase is not normally rate-limiting in cellular respiration, but that, after a period of exposure to CAP-S, an enzyme whose synthesis is inhibited by CAP-S becomes rate-limiting. This enzyme is not cytochrome oxidase, but more likely another component of the respiratory chain. Both CAP and TET inhibited cytochrome oxidase accumulation in non-growing as well as in growing fibroblasts, with enzyme levels declining to 50% of control values in 48 hr; after that time, a new, lower steady state level of enzyme was attained. Oxygen uptake was not inhibited in fibroblasts by short (4-hr) exposure to CAP, CAP-S or TET in concentrations up to 1 mM. Speculation: Antibiotics which act by inhibiting bacterial protein synthesis also inhibit mitochondrial enzyme synthesis in virtually all eukaryotic organisms and cells. The rarity of obvious clinical sequellae in patients receiving these agents is explained by the fact that cytochrome oxidase and other components of the respiratory chain, the synthesis of which is inhibited by these agents, are not normally rate-limiting in cellular respiration. After prolonged exposure to these antibiotics, a rate-limiting level of a component of the respiratory chain is reached, but its activity declines slowly and the restriction in respiratory capacity is consequently modest.

We surveyed 64 individuals with the diagnosis of Brachmann-de Lange syndrome (BDLS) to determine the natural course and cause of the disorder. The 64 individuals were ascertained through membership in a national organization, the Cornelia de Lange Syndrome (CDLS) Foundation, comprised of families who have a relative with BDLS. We surveyed 64 families by questionnaire and personally examined 24 of the 64. Our data suggest that lower birth weight correlates with a more severe phenotype, specifically including severe upper limb malformations and greater psychomotor retardation. The lower birth weight group showed a significant excess of females. The miscarriage rate was normal and there were no recurrences reported in the 64 families we surveyed. Major management problems included feeding problems and projectile vomiting, behavioral problems including frequent tantrums, hearing and dental difficulties, and recurrent respiratory tract infections. The oldest, teenaged subjects in our study entered puberty; although pregnancy has not been reported in the syndrome, it is likely that people with BDLS are fertile. Though most BDLS children reared at home survive through adolescence, a significant degree of psychomotor retardation and difficult medical management problems still occur.

Methods have been optimized for the isolation, growth and passage of differentiating human epidermal cells in dispersed cell culture. Human neonatal foreskin epidermis was separated from dermis after floating the skin on trypsin. Keratinocytes were isolated by dissociation of epidermal sheets in calcium, magnesium-free phosphate buffered saline. Under culture conditions established for mouse keratinocytes, human epidermal cells retained epithelial morphology for 1–2 mo and could be subcultured 2–3 times. Cells exposed to modified culture conditions were tested for growth at low cell density and ability to be repeatedly subcultured while maintaining normal epithelial morphology. Optimal conditions were determined by counting the number of colonies formed 1–3weeks after plating 0.2–1 × 10 4 cells/cm 2 and by measuring the maximum number of passages attainable with cultures plated at 10 5 cells/cm 2 . Compared to tissue culture plastic, a substrate of collagen (prepared by acid extraction of rat tail tendons) increased colony formation 2–4 fold. Lowering the culture temperature from 37° to 34° or 31°C reduced colony number to 50% and 10% respectively. Serum concentrations of 10–20% yielded 2–4 fold more colonies than lower or higher levels. Seven commercial or especially formulated media did not enhance either growth parameter. Both colony formation and number of passages were increased at pH 7.0–7.2 compared to lower or higher pH. Medium ionic calcium concentrations of 0.3–0.4mM doubled the number of colonies and passages compared to higher or lower levels. These improvements in culture conditions enhance the suitability of human keratinocyte cultures for a number of biological studies.

Bullous pemphigoid (BP) antigen is a normal basement membrane zone antigen of epidermis and other stratified squamous epithelia. It is defined immunologically by antibodies in the sera of patients with the subepidermal blistering disease BP. In this study we sought to demonstrate that epidermal cells synthesize this antigen, to determine the immunological specificity of BP antibodies and to characterize this antigen. Cultured human epidermal cells (HEC) and a spontaneously transformed mouse epidermal cell line (Pam) both demonstrated BP antigen by indirect immunofluorescence. To characterize the antigen, these cells were radiolabeled with 35 S-methionine or 14 C-amino acids and extracts were immunoprecipitated using nine different BP sera. Immunoprecipitated proteins were identified using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. All nine BP sera precipitated a protein with disulfide-linked chains of apparent molecular weight approximately 220 kd. Eight normal human sera and six pemphigus vulgaris sera, as well as antibodies directed against fibronectin and laminin, did not precipitate this protein. Furthermore, it was not precipitated by BP sera from radiolabeled extracts of fibroblasts. The protein was soluble in Tris-HCI buffered saline but was not secreted into the culture medium. These studies demonstrate that BP antigen is synthesized by epidermal cells in culture, different patients with BP have antibodies against the same protein, and BP antigen can be identified on SDS-PAGE as a high molecular weight protein consisting of disulfide-linked chains of approximate molecular weight 220 kd.

The major differentiation products of mouse epidermis are keratins of 40-70 kilodaltons (kDal). We have prepared a library of cDNA clones from total poly(A)+ RNA from newborn mouse epidermis. Clones corresponding to the major in vivo keratins of 55, 59, and 67 kDal have been isolated and characterized. By RNA blot analysis of poly(A)+ RNA from newborn mouse epidermis, we have identified RNA species that are approximately 1,600, 2,000, and 2,400 nucleotides in length and are complementary to the cDNAs for the 55-, 59-, and 67-kDal keratins, respectively. Analysis of RNA from primary cultures of newborn mouse epidermis by this same technique shows greatly reduced levels of these RNAs. Transcripts complementary to all three cloned cDNAs are abundant in 14- to 16-day embryonic and adult mouse skin. Thus, altered expression in culture does not appear to be due to induction of a developmentally programmed switch by placing the cells in culture but instead is due to factors modulating expression within the culture system. Because the 55-, 59-, and 67-kDal keratins are the major proteins in epidermis they probably represent keratin associated with terminal differentiation. The expression data suggest that cultured cells are blocked in expression of differentiation keratins but instead synthesize other keratin family members probably related to cytoskeletal functions.

Studies were conducted to determine the effects of the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured human epidermal cells for comparison with known effects on mouse keratinocytes. In contrast to its effect on mouse cells, TPA did not stimulate human epidermal cell DNA synthesis. TPA stimulated differentiation in human keratinocytes resulting in sloughing of many cells by the 3rd day after exposure. Quantitative assays revealed that 50% of the TPA-exposed population was composed of cornified cells as opposed to 8% in untreated controls. A morphologically distinct cell type (TT cell) emerged after TPA treatment which was triangular in shape, did not stratify, appeared to proliferate rapidly and at most TPA concentrations became the predominant cell type within 1–2 weeks. Cultures composed predominantly of TT cells formed few cornified envelopes, grew well in the absence of TPA and formed colonies at low cell input. In contrast to its effect on keratinocytes, TPA enhanced TT colony formation 3–4-fold and decreased the doubling time of TT cells. Studies were performed to determine the origin of TT cells. Immunofluorescent staining indicated that TT cells lacked the keratinocyte antigens keratin, pemphigus and pemphigoid. Tonofilaments and desmosomes were not seen by electron microscopy. The lack of both melanosomes and standard histochemical DOPA oxidase staining indicated that TT cells were probably not of melanocyte origin. Tests used to identify Langerhans cells were negative. Whereas TT cells, as well as dermal fibroblasts, yielded positive immunofluorescence with antibodies to vimentin, TT cells gave a weak histochemical leucine aminopeptidase reaction, while the reaction of fibroblasts exposed to TPA was strong. Treatment of human dermal fibroblasts with TPA did not yield TT cells. The endothelial cell antigen factor VIII-associated protein was absent by immunofluorescence. These results suggest that the primary effect of TPA on cultured human epidermis is to accelerate terminal differentiation in the keratinocyte population and to stimulate growth of an as yet unidentified cell type.

We have developed a procedure for immune selection in an established human lymphoid cell line based on HL-A, the major human histocompatibility locus. After a single brief exposure to selective conditions, HL-A2 variant clones were isolated from an HL-A2/HL-A3 heterozygous line. The variant clones occurred at a frequency of about 1 × 10 -6 . The variant phenotype was stable during prolonged growth in the absence of antiserum after isolation. The variant sublines bound [unk] 1/1000 the HL-A2 antibody per cell as the parent line. Variation was specific in that expression of antigens not selected against was unimpaired. Loss of the chromosome bearing HL-A2 was excluded as the cause of variation because the variants, like the parent line, were heterozygous for phosphoglucomutase (EC 2.7.5.1) determined by structural locus PGM 3 , which is linked to HL-A.