5' strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5' strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle-dependent manner. Depletion of CtIP, Mre11, Exo1 or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of Brca1 does not, suggesting that the role of Brca1 in HR is primarily to promote Rad51 filament formation, not to regulate end resection.

Here we review how GFAP mutations cause Alexander disease. The current data suggest that a combination of events cause the disease. These include: (i) the accumulation of GFAP and the formation of characteristic aggregates, called Rosenthal fibers, (ii) the sequestration of the protein chaperones alpha B-crystallin and HSP27 into Rosenthal fibers, and (iii) the activation of both Jnk and the stress response. These then set in motion events that lead to Alexander disease. We discuss parallels with other intermediate filament diseases and assess potential therapies as part of this review as well as emerging trends in disease diagnosis and other aspects concerning GFAP.

Plectin is a major intermediate filament (IF)-based cytolinker protein that stabilizes cells and tissues mechanically, regulates actin filament dynamics, and serves as a scaffolding platform for signaling molecules. In this study, we show that plectin deficiency is a cause of aberrant keratin cytoskeleton organization caused by a lack of orthogonal IF cross-linking. Keratin networks in plectin-deficient cells were more susceptible to osmotic shock-induced retraction from peripheral areas, and their okadaic acid-induced disruption (paralleled by stress-activated MAP kinase p38 activation) proceeded faster. Basal activities of the MAP kinase Erk1/2 and of the membrane-associated upstream protein kinases c-Src and PKCdelta were significantly elevated, and increased migration rates, as assessed by in vitro wound-closure assays and time-lapse microscopy, were observed. Forced expression of RACK1, which is the plectin-binding receptor protein for activated PKCdelta, in wild-type keratinocytes elevated their migration potential close to that of plectin-null cells. These data establish a link between cytolinker-controlled cytoarchitecture/scaffolding functions of keratin IFs and specific MAP kinase cascades mediating distinct cellular responses.

Budding yeast, like other eukaryotes, carries its genetic information on chromosomes that are sequestered from other cellular constituents by a double membrane, which forms the nucleus. An elaborate molecular machinery forms large pores that span the double membrane and regulate the traffic of macromolecules into and out of the nucleus. In multicellular eukaryotes, an intermediate filament meshwork formed of lamin proteins bridges from pore to pore and helps the nucleus reform after mitosis. Yeast, however, lacks lamins, and the nuclear envelope is not disrupted during yeast mitosis. The mitotic spindle nucleates from the nucleoplasmic face of the spindle pole body, which is embedded in the nuclear envelope. Surprisingly, the kinetochores remain attached to short microtubules throughout interphase, influencing the position of centromeres in the interphase nucleus, and telomeres are found clustered in foci at the nuclear periphery. In addition to this chromosomal organization, the yeast nucleus is functionally compartmentalized to allow efficient gene expression, repression, RNA processing, genomic replication, and repair. The formation of functional subcompartments is achieved in the nucleus without intranuclear membranes and depends instead on sequence elements, protein-protein interactions, specific anchorage sites at the nuclear envelope or at pores, and long-range contacts between specific chromosomal loci, such as telomeres. Here we review the spatial organization of the budding yeast nucleus, the proteins involved in forming nuclear subcompartments, and evidence suggesting that the spatial organization of the nucleus is important for nuclear function.

The desmosomal plaque protein desmoplakin (DP), located at the juncture between the intermediate filament (IF) network and the cytoplasmic tails of the transmembrane desmosomal cadherins, has been proposed to link IF to the desmosomal plaque. Consistent with this hypothesis, previous studies of individual DP domains indicated that the DP COOH terminus associates with IF networks whereas NH2-terminal sequences govern the association of DP with the desmosomal plaque. Nevertheless, it had not yet been demonstrated that DP is required for attaching IF to the desmosome. To test this proposal directly, we generated A431 cell lines stably expressing DP NH2-terminal polypeptides, which were expected to compete with endogenous DP during desmosome assembly. As these polypeptides lacked the COOH-terminal IF-binding domain, this competition should result in the loss of IF anchorage if DP is required for linking IF to the desmosomal plaque. In such cells, a 70-kD DP NH2-terminal polypeptide (DP-NTP) colocalized at cell-cell interfaces with desmosomal proteins. As predicted, the distribution of endogenous DP was severely perturbed. At cell-cell borders where endogenous DP was undetectable by immunofluorescence, there was a striking absence of attached tonofibrils (IF bundles). Furthermore, DP-NTP assembled into ultrastructurally identifiable junctional structures lacking associated IF bundles. Surprisingly, immunofluorescence and immunogold electron microscopy indicated that adherens junction components were coassembled into these structures along with desmosomal components and DP-NTP. These results indicate that DP is required for anchoring IF networks to desmosomes and furthermore suggest that the DP-IF complex is important for governing the normal spatial segregation of adhesive junction components during their assembly into distinct structures.

Intermediate filaments are assembled from a diverse group of evolutionary conserved proteins and are specified in a tissue-dependent, cell type-dependent, and context-dependent fashion in the body. Genetic mutations in intermediate filament proteins account for a large number of diseases, ranging from skin fragility conditions to cardiomyopathies and premature aging. Keratins, the epithelial-specific intermediate filaments, are now recognized as multi-faceted effectors in their native context. In this review, we emphasize the recent progress made in defining the role of keratins towards the regulation of cytoarchitecture, cell growth and proliferation, apoptosis, and cell motility during embryonic development, in normal adult tissues, and in select diseases such as cancer.

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.

Glial fibrillary acidic protein (GFAP) is the principle intermediate filament (IF) protein in astrocytes. Mutations in the GFAP gene lead to Alexander disease (AxD), a rare, fatal neurological disorder characterized by the presence of abnormal astrocytes that contain GFAP protein aggregates, termed Rosenthal fibers (RFs), and the loss of myelin. All GFAP mutations cause the same histopathological defect, i.e. RFs, though little is known how the mutations affect protein accumulation as well as astrocyte function. In this study, we found that GFAP accumulation induces macroautophagy, a key clearance mechanism for prevention of aggregated proteins. This autophagic response is negatively regulated by mammalian target of rapamycin (mTOR). The activation of p38 MAPK by GFAP accumulation is in part responsible for the down-regulation of phosphorylated-mTOR and the subsequent activation of autophagy. Our study suggests that AxD mutant GFAP accumulation stimulates autophagy, in a manner regulated by p38 MAPK and mTOR signaling pathways. Autophagy, in turn, serves as a mechanism to reduce GFAP levels.

Research on the cell fate determination of embryonic stem cells is of enormous interest given the therapeutic potential in regenerative cell therapy. Human embryonic stem cells (hESCs) have the ability to renew themselves and differentiate into all three germ layers. The main focus of this study was to examine factors affecting derivation and further proliferation of multipotent neuroepithelial (NEP) cells from hESCs. hESCs cultured in serum-deprived defined medium developed distinct tube structures and could be isolated either by dissociation or adherently. Dissociated cells survived to form colonies of cells characterized as NEP when conditioned medium from human hepatocellular carcinoma HepG2 cell line (MEDII) was added. However, cells isolated adherently developed an enriched population of NEP cells independent of MEDII medium. Further characterization suggested that they were NEP cells because they had a similar phenotype profile to in vivo NEP cells and expression SOX1, SOX2, and SOX3 genes. They were positive for Nestin, a neural intermediate filament protein, and Musashi-1, a neural RNA-binding protein, but few cells expressed further differentiation markers, such as PSNCAM, A2B5, MAPII, GFAP, or O4, or other lineage markers, such as muscle actin, alpha fetoprotein, or the pluripotent marker Oct4. Further differentiation of these putative NEP cells gave rise to a mixed population of progenitors that included A2B5-positive and PSNCAM-positive cells and postmitotic neurons and astrocytes. To proliferate and culture these derived NEP cells, ideal conditions were obtained using neurobasal medium supplemented with B27 and basic fibroblast growth factor in 5% oxygen. NEP cells were continuously propagated for longer than 6 months without losing their multipotent cell characteristics and maintained a stable chromosome number.

Nuclear lamins are type V intermediate filament proteins. They are the major building blocks of the peripheral nuclear lamina, a complex meshwork of proteins underlying the inner nuclear membrane. In addition to providing nuclear shape and mechanical stability, they are required for chromatin organization, transcription regulation, DNA replication, nuclear assembly and nuclear positioning. Over the past few years, interest in the lamins has increased because of the identification of at least 12 distinct human diseases associated with mutations in the LMNA gene, which encodes A-type lamins. These diseases, collectively termed laminopathies, affect muscle, adipose, bone, nerve and skin cells and range from muscular dystrophies to accelerated aging.

The mammalian cell cytoskeleton consists of a diverse group of fibrillar elements that play a pivotal role in mediating a number of digestive and nondigestive cell functions, including secretion, absorption, motility, mechanical integrity, and mitosis. The cytoskeleton of higher-eukaryotic cells consists of three highly abundant major protein families: microfilaments (MF), microtubules (MT), and intermediate filaments (IF), as well as a growing number of associated proteins. Within digestive epithelia, the prototype members of these three protein families are actins, tubulins, and keratins, respectively. This review highlights the important structural, regulatory, functional, and unique features of the three major cytoskeletal protein groups in digestive epithelia. The emerging exciting biological aspects of these protein groups are their involvement in cell signaling via direct or indirect interaction with a growing list of associated proteins (MF, MT, IF), the identification of several disease-causing mutations (IF, MF), the functional role that they play in protection from environmental stresses (IF), and their functional integration via several linker proteins that bridge two or potentially all three of these groups together. The use of agents that target specific cytoskeletal elements as therapeutic modalities for digestive diseases offers potential unique areas of intervention that remain to be fully explored.

Localization of vinculin at the sarcolemma of striated muscle fibers defines an orthogonal lattice. The costameres of the lattice are the riblike bands of vinculin that run perpendicular to the long axis of the fiber, repeat in register with I bands of the subjacent myofibrils, and seem to couple the myofibril to the sarcolemma [Pardo et al 1982, 1983a]. The colocalization studies presented in this paper show that gamma actin, spectrin, and intermediate filament antigens are additional components of this lattice of costameres. In addition, the results show that gamma actin and spectrin are also components of the internal network of collars, first visualized with antibody to desmin [Granger and Lazarides, 1978], that connects the myofibrils to each other at the level of the Z line.

Reproducible multi-stage progression to invasive squamous carcinoma of the epidermis has been achieved in transgenic mice expressing the HPV16 early-region genes, including the E6/E7 oncogenes, under the control of the human keratin-14 promoter/enhancer. Although 100% of K14-HPV16 transgenic animals develop hyperplastic and/or dysplastic lesions in several inbred backgrounds, including C57BL/6, BALB/c, and SSIN/SENCAR, only mice backcrossed into the FVB/n background progress to malignant squamous cell carcinomas of two pathological grades, well differentiated and moderate/poorly differentiated (WDSC or MPDSC, respectively), each displaying characteristic patterns of malignant behavior. WDSCs typically arise within the epidermis of the ear and invade deeply into the underlying dermis but fail to metastasize, whereas MPDSCs develop on the chest and truncal skin and invariably metastasize to regional lymph nodes. The transition to the malignant state, in 21% of FVB/n transgenic mice, is characterized by alteration of the repertoire of keratin intermediate filament proteins expressed within neoplastic epidermis, such that WDSCs maintain expression of keratins common to terminally differentiating stratified keratinocytes (K10), whereas MPDSCs are distinguished from WDSCs by activation of embryonic and mucosal keratins (K13, K8, and K19). Precursor hyperplastic and dysplastic lesions are characterized by a progressively increased proliferative index, striking morphological alterations in keratinocyte cell-cell and cell-matrix interactions, and extensive remodeling of the underlying dermal stroma. Remarkably, this extensive stromal remodeling, which may facilitate both angiogenesis and eventual tumor cell invasion, develops early at the dysplastic stage in all animals well before malignant conversion.

Nucleoprotein complexes containing viral DNA and cellular histones were extracted from nuclei of permissive cells infected with polyoma virus or simian virus 40 (SV40) and examined by electron microscopy. Polyoma and SV40 nucleoprotein complexes are almost identical. They appear as relaxed circular molecules consisting of 20 to 21 globular particles interconnected by thin filaments. Their contour length in 0.02 M salt is 2.7 times shorter than that of viral DNA form I obtained after dissociation of the proteins in 1 M NaCl. The nucleosomes have an average diameter of 12.5 nm. Each nucleosome contains 175 to 205 DNA base pairs condensed fivefold in length. The nucleosomes are regularly spaced on the circular molecule. The internucleosomal filaments are made of naked DNA, and each filament contains about 55 base pairs. The partial sensitivity of the nucleoprotein complex to cleavage by EcoR1 endonuclease suggests that the nucleosomes are not formed at specific sites on the viral genome. Faster sedimenting nucleoprotein complexes containing replicative intermediates were studied. Isopycnic centrifugation in metrizamide gradients in the absence of aldehyde fixation showed that these molecules conserved the same DNA-to-protein ratio as the form I DNA-containing complexes.

We have previously observed that breast cancer cell lines could exhibit either epithelial or fibroblastic phenotypes as reflected by their morphologies and intermediate filament protein expression (C. L. Sommers, D. Walker-Jones, S. E. Heckford, P. Worland, E. Valverius, R. Clark, M. Stampfer, and E. P. Gelmann, Cancer Res., 49:4258-4263, 1989). Fibroblastoid, vimentin-expressing breast cancer cell lines are more invasive in vitro and in vivo (E. W. Thompson, S. Paik, N. Brunner, C. L. Sommers, G. Zugmaier, R. Clarke, T. B. Shima, J. Torri, S. Donahue, M. E. Lippman, G. R. Martin, and R. B. Dickson, J. Cell. Physiol., 150: 534-544, 1992). We hypothesized that a breast cancer cell with an epithelial phenotype could undergo a transition to a fibroblastic phenotype, possibly resulting in more invasive capacity. We now show that two Adriamycin-resistant MCF-7 cell lines and a vinblastine-resistant ZR-75-B cell line have undergone such a transition. Adriamycin-resistant MCF-7 cells express vimentin, have diminished keratin 19 expression, have lost cell adhesion molecule uvomorulin expression, and have reduced formation of desmosomes and tight junctions as determined by reduced immunodetection of their components desmoplakins I and II and zonula occludens (ZO)-1. Other MCF-7 cell lines selected for resistance to vinblastine and to Adriamycin and verapamil did not have these characteristics, indicating that drug selection does not invariably cause these phenotypic changes. In addition, to determine if vimentin expression in MCF-7 cells alone could manifest a fibroblastic phenotype, we transfected the full-length human vimentin complementary DNA into MCF-7 cells. Although vimentin expression was achieved in MCF-7 cells, it did not affect the phenotype of the cells in terms of the distribution of keratins, desmoplakins I and II, ZO-1, or uvomorulin or in terms of in vitro invasiveness. We conclude that vimentin expression is a marker for a fibroblastic and invasive phenotype in breast cancer cells but does not by itself give rise to this phenotype.

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.

Recent studies have characterised a family of giant cytoskeletal crosslinkers encoded by the short stop gene in Drosophila and the dystonin/BPAG1 and MACF1 genes in mammals. We refer to the products of these genes as spectraplakins to highlight the fact that they share features with both the spectrin and plakin superfamilies. These genes produce a variety of large proteins, up to almost 9000 residues long, which can potentially extend 0.4 micro m across a cell. Spectraplakins can interact with all three elements of the cytoskeleton: actin, microtubules and intermediate filaments. The analysis of mutant phenotypes in BPAG1 in mouse and short stop in Drosophila demonstrates that spectraplakins have diverse roles. These include linking the plasma membrane and the cytoskeleton, linking together different elements of the cytoskeleton and organising membrane domains.

Cytolinkers are giant proteins that can stabilize cells by linking actin filaments, intermediate filaments, and microtubules (MTs) to transmembrane complexes. Dystrophin is functionally similar to cytolinkers, as it links the multiple components of the cellular cytoskeleton to the transmembrane dystroglycan complex. Although no direct link between dystrophin and MTs has been documented, costamere-associated MTs are disrupted when dystrophin is absent. Using tissue-based cosedimentation assays on mice expressing endogenous dystrophin or truncated transgene products, we find that constructs harboring spectrinlike repeat 24 through the first third of the WW domain cosediment with MTs. Purified Dp260, a truncated isoform of dystrophin, bound MTs with a K(d) of 0.66 microM, a stoichiometry of 1 Dp260/1.4 tubulin heterodimer at saturation, and stabilizes MTs from cold-induced depolymerization. Finally, alpha- and beta-tubulin expression is increased approximately 2.5-fold in mdx skeletal muscle without altering the tubulin-MT equilibrium. Collectively, these data suggest dystrophin directly organizes and/or stabilizes costameric MTs and classifies dystrophin as a cytolinker in skeletal muscle.

Human endothelial cells prepared from unbilical cords are characterized in parallel by electron microscopy and indirect immunofluorescence microscopy using specific antibodies against different classes of intermediate-sized filaments. The strongly developed, loose bundles of intermediate-sized filaments typically found in these cells are not decorated by antibodies against prekeratin or antibodies against smooth muscle desmin. They are, however, strongly decorated by antibodies directed against murine "vimentin," i.e., the 57,000 mol wt polypeptide which is the major protein of the intermediate-sized filaments predominant in various cells of mesenchymal origin. Cytoskeletal preparations greatly enriched in intermediate-sized filaments show the enrichment of a polypeptide band comigrating with murine vimentin. This shows that the intermediate-sized filaments that are abundant in human endothelial cells are predominantly of the vimentin type and can be demonstrated by their cross-reaction with the vimentin of rodents. These data also strengthen the evidence for several subclasses of intermediate-sized filaments, which can be distinguished by immunological procedures.

Neoplastic cells are often characterized by specific morphological abnormalities of the nuclear envelope (NE), which have been used for cancer diagnosis for more than a century. The NE is a double phospholipid bilayer that encapsulates the nuclear genome, regulates all nuclear trafficking of RNAs and proteins and prevents the passive diffusion of macromolecules between the nucleoplasm and the cytoplasm. Whether there is a consequence to the proper functioning of the cell and loss of structural integrity of the nucleus remains unclear. Using live cell imaging, we characterize a phenomenon wherein nuclei of several proliferating human cancer cell lines become temporarily ruptured during interphase. Strikingly, NE rupturing was associated with the mislocalization of nucleoplasmic and cytoplasmic proteins and, in the most extreme cases, the entrapment of cytoplasmic organelles in the nuclear interior. In addition, we observed the formation of micronuclei-like structures during interphase and the movement of chromatin out of the nuclear space. The frequency of these NE rupturing events was higher in cells in which the nuclear lamina, a network of intermediate filaments providing mechanical support to the NE, was not properly formed. Our data uncover the existence of a NE instability that has the potential to change the genomic landscape of cancer cells.

The time course of loss of the 55,000-Da intermediate filament protein desmin was measured in rabbit muscles subjected to cyclic eccentric contraction. Rabbit extensor digitorum longus (EDL) and tibialis anterior (TA) muscles were examined 5 or 15 min after eccentric exercise and 1 h or 1 day after 30 min of an eccentric exercise protocol (n = 16 rabbits). The earliest change noted was a significant loss of desmin labeling in 2.5 +/- 0.63% of the rabbit EDL muscle fibers (P < 0.005) 5 min after initiation of eccentric exercise. Some loss of TA fiber desmin was also apparent at this time period (0.24 +/- 0.19%), but the magnitude was not significantly different from zero (P>> 0.2). Fifteen minutes after initiation of exercise, desmin loss was more pronounced, increasing to 7.4 +/- 1.4 and 4.6 +/- 1.0% in the EDL and TA, respectively (P < 0.005). Finally, 1 day after 30 min of eccentric exercise, the percentage of fibers without desmin staining rose to 23.4 +/- 3.7 and 7.7 +/- 2.4% in the EDL and TA, respectively (P < 0.001). Loss of desmin staining occurred in the absence of contractile or metabolic protein disruption. Increased staining intensity of the intrasarcomeric cytoskeletal protein titin and an inability to exclude plasma fibronectin were also observed in most but not all fibers that had lost desmin staining. Desmin disruption thus represents a very early structural manifestation of muscle injury during eccentric contraction. Cytoskeletal disruption may predispose the contractile apparatus to previously reported structural damage.

The intermediate filament (IF) system of the various cells of human, pig and rat ovaries was studied by electron microscopy, by immunolocalization using antibodies to cytokeratins, vimentin, desmin and desmoplakin, and by two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissue samples. In human ovaries, surface epithelial cells (mesothelium) were stained by antibodies against cytokeratins, desmoplakins and vimentin. Biochemical analysis revealed cytokeratins Nos. 8, 18 and 19, together with variable amounts of No. 7. Granulosa cells of follicles of all stages were also positive for cytokeratins, desmoplakins and vimentin, in agreement with the electron microscopic finding of desmosomes in these cells. As the follicle matured, the cytokeratin content usually appeared to decrease, whereas vimentin remained unchanged. On gel electrophoresis, granulosa cells presented cytokeratins Nos. 8 and 18 and vimentin. Rete ovarii cells were also positive for both cytokeratins, desmoplakins and vimentin, and the electron microscopy revealed numerous desmosome-tonofilament complexes. Oocytes appeared to be devoid of IFs. Corpus luteum cells were rich in vimentin but biochemical analysis also revealed small amounts of cytokeratins Nos. 8 and 18. In contrast, cells of the ovarian stroma and luteinized stromal nodules were positive for vimentin only. A certain type of scattered stromal cells, especially around tertiary follicles and corpora lutea, and also desmin-positive. Pig and rat ovaries differed from human ones in that vimentin was not detected in ovarian mesothelium and cytokeratins were not seen in granulosa cells. The latter, however, contained significant amounts of vimentin. These results indicate that three cell types of human ovary, i.e. surface epithelial, granulosa and rete ovarii cells, can be regarded as true epithelial cells which, however, simultaneously express vimentin, a phenomenon frequently seen in cultured epithelial cells but uncommon in epithelial tissues. The presence of cytokeratins in granulosa cells in all types of human follicles is discussed with regard to the development of these cells. In contrast, granulosa cells of the other two mammalian species only display vimentin IF. Such differences between different mammalian species in IF composition of ovarian components present an example which precludes extrapolation of data from one species to another. The results are discussed in relation to current views of the histogenesis of various ovarian tumors.

Using a cloned cDNA complementary to a portion of the mRNA for the 50 kd human epidermal keratin, we have screened a human genomic library and have isolated and sequenced the gene encoding this keratin. A comparison of the keratin gene with the very distantly related vimentin gene has enabled us to explore the relation between the evolutionary conservation of structure in intermediate filament (IF) subunits and the conservation of structure in IF genes. Our results reveal that not only the secondary structure of the IF proteins, but also the structural skeleton of their genes, has been maintained throughout evolution. These characteristics have persisted despite considerable flexibility in both protein and nucleic acid sequence. Surprisingly, although the positions of the introns within these two genes are highly conserved, they do not seem to correspond to the boundaries of the structural domains common to all IF subunits.