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We describe the chromosomal organization of the major oocyte and somatic 5S RNA genes of Xenopus laevis in chromatin isolated from erythrocyte nuclei. Both major oocyte and somatic 5S DNA repeats are associated with nucleosomes; however, differences exist in the organization of chromatin over the oocyte and somatic 5S RNA genes. The repressed oocyte 5S RNA gene is protected from nuclease digestion by incorporation into a nucleosome, and the entire oocyte 5S DNA repeat is assembled into a loosely positioned array of nucleosomes. In contrast, the potentially active somatic 5S RNA gene is accessible to nuclease digestion, and the majority of somatic 5S RNA genes appear not to be incorporated into (38). The association of histone Hi with the oocyte genes may be involved in actually establishing repression (28, 49). However, the relationship between the organization of the oocyte genes into nucleosomes, the presence or absence of histone Hi within nucleosomal DNA containing the oocyte genes, and the repressed state of the oocyte genes in vivo has not yet been resolved.In contrast to the existing analyses of the chromosomal organization of the major oocyte 5S RNA genes, no comparable study of the somatic 5S RNA genes has appeared. In this report, we have examined the chromosomal organization of both the oocyte and the somatic 5S RNA genes in erythrocytes. We find that the oocyte genes are repressed and that the whole oocyte 5S DNA repeat is organized into a positioned array of nucleosomes. Consistent with earlier work (21, 56), the internal promoter elements of the major oocyte 5S RNA gene are incorporated into a nucleosome.* Corresponding author.Disruption of this chromatin organization occurs following the removal of histone Hi. This facilitates the activation of transcription of 5S RNA genes in a complete transcription extract. Readdition of histone Hi to this active chromatin selectively represses transcription of the oocyte genes and alters the chromatin structure over the oocyte genes. In contrast, the somatic 5S DNA repeats are assembled into arrays of nucleosomes that do not appear to have any uniform organization with respect to DNA sequence. We present evidence for a region around the somatic 5S RNA gene that is accessible to nucleases and for the association of transcription factors with both the internal control region (ICR) and the 5' flanking region of this gene. MATERIALS AND METHODSPreparation of chromatin. X. laevis females were purchased from Xenopus I. Blood was collected from the heart into 1 x STM (90 mM NaCl, 10 mM Tris, 2 mM MgCI2, pH 7.4)-heparin (10 U/ml) on ice. Erythrocytes were washed twice in the same buffer by centrifugation at 600 x g for 5 min (26,56). Hepatocytes were isolated from Xenopus liver (39).Nuclei were prepared from erythrocytes and hepatocytes by established methods (23,50). Cells were Dounce homogenized, and the nuclei were resuspended in 50% glycerol-50 mM Tris HCI (pH 7.5)-100 mM KCI-5 mM MgCl2-2 mM dithiothreitol (DTT)-0.5 mM phenylmethylsulfonyl fluoride (PMSF)-0.1% ...

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The most frequent mutation that causes the autosomal dominant skin disease epidermolytic hyperkeratosis (EHK) is an arginine to histidine substitution at position 10 in the 1A segment of the rod domain of keratin 10. As an initial step toward developing a strategy for treating EHK, a cell line, EH18-1, was established after keratinocytes derived from an EHK patient with this mutation were immortalized by a recombinant retrovirus encoding the E6 and E7 genes of human papillomavirus type 18. EH18-1 cells synthesize considerable amounts of keratin 10 mRNA and protein when maintained in either submerged cultures or in organotypic cultures. When grown in organotypic culture, EH18-1 cells form multiple layers and express keratin 10 and filaggrin predominantly in the upper layers. Thus, the EH18-1 cell line exhibits several morphological and biochemical markers of terminal epidermal differentiation. A semiquantitative reverse transcriptase polymerase chain reaction assay for keratin 10 mRNA was developed to distinguish between expression of the normal and the mutant alleles. The EH18-1 keratinocyte cell line will be useful in developing protocols for gene therapy of EHK that may be monitored by reverse transcriptase polymerase chain reaction of either allele.

Profilaggrin is a major highly phosphorylated protein component of the keratohyalin granules of mammalian epidermis. It contains 10 to 12 tandemly repeated filaggrin units and is processed into the intermediate filament-associated protein filaggrin by specific dephosphorylation and proteolysis during terminal differentiation of the epidermal cells. Later, filaggrin itself is degraded to free amino acids that participate in maintenance of epidermal flexibility. The present paper describes the structural organization of the 5' region of the human profilaggrin gene as well as the amino terminus of the profilaggrin protein. The primary profilaggrin transcript consists of three exons and two introns. The first exon (exon I) is only 54 bp and is untranslated. The coding sequences are distributed between exon II (159 bp) and exon III, which contains the information for 10 to 12 filaggrin repeats (972 bp each) and the 3' noncoding sequences. A very large intron separates exons I and II. The combination of a very short exon I with an unusually long intron 1 makes the structure of the profilaggrin gene unique among the epidermally expressed genes investigated so far. Comparison of the expression patterns revealed by primer extension and RNase protection analysis of foreskin epidermal and cultured keratinocyte RNAs suggests that alternately spliced messages, which are different from profilaggrin mRNA, are transcribed from the profilaggrin gene system at earlier stages of epidermal differentiation. The amino terminus of profilaggrin exhibits a significant homology to the small calcium-binding S100-like proteins. It contains two alpha-helical regions, termed EF-hands, that bind calcium in vitro. This is the first example of functional calcium-binding domains fused to a structural protein. We suggest that in addition to its role in filament aggregation and the maintenance of epidermal flexibility, profilaggrin may play an important role in the differentiation of the epidermis by autoregulating its own processing in a calcium-dependent manner or by participating in the transduction of calcium signal in epidermal cells.

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We describe the chromosomal organization of the major oocyte and somatic 5S RNA genes of Xenopus laevis in chromatin isolated from erythrocyte nuclei. Both major oocyte and somatic 5S DNA repeats are associated with nucleosomes; however, differences exist in the organization of chromatin over the oocyte and somatic 5S RNA genes. The repressed oocyte 5S RNA gene is protected from nuclease digestion by incorporation into a nucleosome, and the entire oocyte 5S DNA repeat is assembled into a loosely positioned array of nucleosomes. In contrast, the potentially active somatic 5S RNA gene is accessible to nuclease digestion, and the majority of somatic 5S RNA genes appear not to be incorporated into positioned nucleosomes. Evidence is presented supporting the stable association of transcription factors with the somatic 5S RNA genes. Histone H1 is shown to have a role both in determining the organization of nucleosomes over the oocyte 5S DNA repeat and in repressing transcription of the oocyte 5S RNA genes.

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