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Insulin-like growth factors (IGFs) play an important role in tumour growth and development. We hypothesized that this is also the case for medulloblastomas, which are highly malignant cerebellar brain tumours usually occurring in children. In these tumours the expression patterns of IGF-I and -II mRNA were studied. Tumour specimens obtained from 12 children and two adults at diagnosis were hybridized in situ with digoxigenin-labelled cRNA probes for hIGF-I and hIGF-II mRNAs. In all cases, tumour cells showed abundant expression of IGF-I mRNA. Nine of the 14 tumours showed variable but significant IGF-II expression. In these tumours, the hybridization signal almost exclusively colocalized with a subpopulation of Ki-M1P positive cells that were identified as ramified microglia (RM) cells. In the five tumours without IGF-II expression, microglia/brain macrophages with a more rounded amoeboid-like morphology predominated. RM cells in normal cerebellar tissues, residing abundantly in areas of the white and, to a less extent, in the grey matter, were IGF-II mRNA-negative. These RM cells showed a thinner and more extensively branched appearance and were more evenly distributed than those encountered in medulloblastoma. Probably, during the transformation from the resting ramified towards the amoeboid morphology (or vice versa) IGF-II mRNA expression is only temporarily induced. The physiological meaning of the induction of IGF-II mRNA expression by these cells in medulloblastoma remains unclear but any IGF-II peptide synthesized could exert unfavourable mitogenic and antiapoptotic effects on adjacent tumour cells. However, in this relatively small number of cases we could not find any indications for a relationship between clinical characteristics of the various cases and the extent of IGF-II mRNA expression.

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The insulin-like growth factors, IGF-I and IGF-II, and their binding proteins play an important role in the growth and development of the central nervous system. In the brain, colocalization of IGFs and IGFBPs often occurs, suggesting that IGFBPs can modulate IGF action. In one strain of our human (h)IGF-II transgenic mice, which carry an hIGF-II transgene driven by the H-2Kb promoter, we found overexpression of hIGF-II in the brain, as measured by Northern blot analysis. To clarify the localization and influence of the hIGF-II transgene on different components of the GH-IGF axis in the brain, we studied the expression pattern of the hIGF-II transgene, endogenous IGF-I and IGF-II, and IGFBP-2, -3 and -5 in the brain of prepubertal 4-week-old mice, using nonradioactive in situ hybridization. We found that the hIGF-II transgene is exclusively expressed in neurons of the piriform cortex, the cerebral cortex, the medulla oblongata and the granular layer of the cerebellum. In general, this pattern is comparable to the expression pattern of endogenous IGF-I, with a few exceptions: there is no expression of IGF-I in the granular layer of the cerebellum, whereas the Purkinje cells of the cerebellum and thalamus both express IGF-I but no hIGF-II transgene. This hIGF-II transgene expression pattern contrasts markedly with endogenous IGF-II expression, which is mainly located in nonneuronal cells such as the meninges and choroid plexus, and in some nuclei of the medulla oblongata. The hIGF-II transgene affects neither endogenous IGF-I and IGF-II expression, nor the expression of IGFBP-3, which is located in the choroid plexus. Although the hIGF-II transgene is expressed in neuronal structures similar to IGF-I and IGFBP-5, it is not able to regulate IGFBP-5 expression, as has previously been reported for IGF-I. In the medulla oblongata, the IGFBP-2 expression level showed 10-fold upregulation by the transgene, suggesting a modulating role for IGFBP-2 at the hIGF-II transgene action in this region.

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Glucocorticoid (GC) treatment in childhood can lead to suppression of longitudinal growth as a side effect. The actions of GCs are thought to be mediated in part by impaired action of the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins (IGFBP-1 to -6). We have studied the effects of GCs on IGF and IGFBP expression at the local level of the growth plate, using non-radioactive in situ hybridization. We treated 3-week-old normal mice for 4 weeks with dexamethasone (DXM). We also treated human IGF-II (hIGF-II) transgenic mice in order to investigate whether IGF-II could protect against the growth retarding effect of this GC. DXM treatment resulted in general growth retardation in both mice strains, however, only in normal mice was tibial length decreased. In both normal and hIGF-II trangenic mice, the total width of the growth plate was not affected, whereas the width of the proliferative zone decreased as a result of the DXM treatment. Additionally, only in normal mice, the width of the hypertrophic zone thickened. Only expression of IGF-I, IGF-II and IGFBP-2 could be detected in the growth plates of 7-week-old normal mice. IGFBP-1, -3, -4, -5 and -6 mRNAs were not detected. DXM treatment of normal mice induced a significant 2.4-fold increase in the number of cells expressing IGF-I mRNA, whereas IGF-II and IGFBP-2 mRNA levels were not affected. In hIGF-II transgenic mice, IGF-I mRNA levels were significantly increased, while endogenous IGF-II and IGFBP-2 mRNAs were unaffected, compared to normal animals. DXM treatment of the hIGF-II transgenic mice induced a further increase of IGF-I mRNA expression, to a similar extent as in DXM-treated normal mice. The increase of IGF-I due to DXM treatment in normal mice might be a reaction in order to minimize the GC-induced growth retardation. Another possibility could be that the increase of IGF-I would contribute to the GC-induced growth retardation by accelerating the differentiation of chondrocytes, resulting in accelerated ossification. In the growth plates of hIGF-II transgenic mice, the higher basal level of IGF-I, might be responsible for the observed partial protection against the adverse effects of GCs on bone.

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High (pharmacological) doses of glucocorticoids inhibit the proliferation of growth plate chondrocytes, which leads to one of the side-effects of these steroids, namely suppression of longitudinal growth. Growth inhibition by glucocorticoids is thought to be mediated in part by impaired action of components of the IGF axis, which are important for chondrocyte regulation and hence for longitudinal growth. The aim of the present study was to determine whether glucocorticoid-induced growth retardation involves changes in IGF axis components. Chondrocytes were isolated from epiphyseal growth plates of neonatal piglets and treated with pharmacological doses of dexamethasone (DXM) for 24 h to study glucocorticoid-induced growth retardation. Under IGF-I-supplemented (10 nM) culture conditions, IGF-binding proteins (IGFBPs)-2, -4 and -5 were secreted by the growth plate chondrocytes and IGFBP-2 protein and mRNA levels were decreased by the DXM treatment, whereas IGFBP-4 and -5 were not affected. Proliferation of the chondrocytes, as measured by [(3)H]thymidine incorporation, was 3.5-fold higher in serum-supplemented medium in contrast to IGF-I-supplemented (10 nM) medium. In the presence of serum, DNA synthesis was significantly inhibited by 50-63% when treated with 100 nM DXM, which was prevented by the glucocorticoid-receptor antagonist Org34116. mRNA levels of IGF axis components were determined using Northern blot analysis. IGFBP-2 to -6 were expressed in the chondrocytes, IGFBP-1 was absent and both IGF-I and IGF-II, and the type I and type II IGF receptors were expressed. Treatment with DXM (100 nM) resulted in a 2-fold increase in mRNA levels of both IGFBP-5 and the type I IGF receptor, whereas IGFBP-2 mRNA levels decreased by 55%, in concert with the decrease in protein level observed under IGF-I-supplemented culture conditions. The changes in mRNA levels due to the DXM treatment were prevented by the glucocorticoid receptor antagonist. Our data show that exposure to pharmacological doses of DXM results in inhibition of proliferation and changes in components of the IGF axis, IGFBP-2 and -5 and the type I IGF receptor, suggesting a role for these components in glucocorticoid-induced growth retardation at the local level of the growth plate.

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Insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is generally believed to inhibit IGF action in the circulation. In contrast, IGFBP-1 has been reported to interact with cell surfaces and enhance IGF-I action locally in some tissues. Renal IGFBP-1 levels are found elevated in various conditions characterized by renal growth (e.g. diabetes mellitus, hypokalemia). To test whether IGFBP-1 is a renotropic factor, IGFBP-1 was administered alone or in combination with IGF-I to Snell dwarf mice, an in vivo model without compensatory feedback effects on growth hormone (GH) secretion. In three control groups of Snell dwarf mice, placebo, GH or IGF-I was administered. Compared with placebo, kidney weight increased in all treated groups, however, with different effects on kidney morphology. Administration of IGF-I, alone or in combination with IGFBP-1, tended to increase glomerular volume, while no changes were seen in the other groups. Administration of IGFBP-1 or IGFBP-1+IGF-I both caused dilatation of the thin limbs of Henle's loop, while GH or IGF-I administration had no visible effect. Furthermore, IGF-I administration resulted in an increased mean number of nuclei per cortical area and renal weight, whereas GH, IGF-I+IGFBP-1 or IGFBP-1 caused a decreased renal nuclei number. In situ hybridization and immunohistochemistry showed specific changes of the renal IGF system expression patterns in the different groups. Particularly, IGFBP-1 administration resulted in extensive changes in the mRNA expression of the renal IGF system, whereas the other administration regimen resulted in less prominent modifications. In contrast, administration of IGFBP-1 and IGFBP-1+IGF-I resulted in identical changes in the protein expression of the renal IGF system. Our results indicate that IGFBP-1, alone or in combination with IGF-I, demonstrated effects on the renal tubular system that differ from the effects of IGF-I.

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The actions of insulin-like growth factor-I (IGF-I) are modulated by IGF binding proteins (IGFBPs). The effects of IGFBP-1 in vivo are insufficiently known, with respect to inhibitory or stimulatory actions on IGF-induced growth of specific organs. Therefore, we studied the effects of IGFBP-1 on IGF-I-induced somatic and organ growth in pituitary-deficient Snell dwarf mice. Human GH, IGF-I, IGFBP-1, and a preequilibrated combination of equimolar amounts of IGF-I and IGFBP-1 were administered sc during 4 weeks. Treatment with IGF-I alone induced a significant increase in body length (108% of control) and weight (112%) as well as an increase in weight of the submandibular salivary glands (135%), kidneys (124%), femoral muscles (111%), testes (129%), and spleen (126%) compared with saline-treated controls. IGFBP-1 alone induced a significant increase in weight of the kidneys (152% of control). Coadministration of IGF-I with IGFBP-1 neutralized the stimulating effects of IGF-I on body length and weight as well as on the femoral muscles and testes. In contrast, the weights of the submandibular salivary glands (143%) were not significantly different from those of IGF-I-treated animals, whereas the weights of the kidneys (171%) and spleen (156%) were significantly increased compared with IGF-I-treated mice. The effect of IGFBP-1 plus IGF-I on kidney weight was not significantly greater than the effect of IGFBP-1 alone. Western ligand blotting showed induction of the IGFBP-3 doublet as well as IGFBPs with molecular masses of 24 kDa, most probably IGFBP-4, by human GH, IGF-I alone, and IGF-I in combination with IGFBP-1. Our data show that coadministration of IGFBP-1 inhibits IGF-I-induced body growth of GH-deficient mice but significantly stimulates the growth promoting effects of IGF-I on the kidneys and the spleen. These data warrant further investigation because differences in concentrations of IGFBP-1 occurring in vivo may influence IGF-I-induced anabolic processes.

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Protein C inhibitor (PCI) is a heparin binding serine protease inhibitor in plasma, which exerts procoagulant activity by inhibiting thrombomodulin-bound thrombin or activated protein C (APC). Since the role of PCI in vivo is largely unknown we generated genetically modified mice with expression of human PCI mRNA in hepatocytes only. Three transgenic lines have been characterized. Transgenic mice did not show gross developmental abnormalities. Two lines showed a pericentral and one line showed a periportal expression pattern of human PCI mRNA in the liver. Genetically modified mice secreted a functional transgenic protein into the circulation (3-5 microg/ml plasma in heterozygous mice and 10 microg/ml in homozygous mice), which inhibited human APC activity in the presence of heparin. Interestingly, transgenic mice in which human PCI was expressed periportally in the liver had the highest specific activity. Endogenous mouse PCI mRNA could only be detected in the male and female reproductive system, but not in the liver, indicating that endogenous PCI levels in the circulation are low or even absent in mice. These results demonstrate that the human PCI transgenic mice are a suitable model for studying the in vivo role of PCI in blood coagulation.

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Overexpression of human insulin-like growth factor II (IGF-II) in transgenic mice does not result in increased overall body growth. The IGF-II overexpression, however, specifically causes growth of the thymus and not of the spleen. We address the question whether the observed differences in growth induction in lymphoid tissues by IGF-II can be related to differences in local IGF binding protein (IGFBP) production, using nonradioactive in situ hybridization and Northern blot analysis. IGFBP-2, -4, and -5 are expressed in both lymphoid tissues of normal mice. The spleen additionally expresses IGFBP-3 and IGFBP-6. IGFBP-1 expression was not detected. Although the expression pattern of the IGFBPs did not change upon IGF-II overexpression, the level of expression changed in a specific manner for each IGFBP. In both the thymus and the spleen of transgenic mice, IGFBP-2 and -5 gene expression was slightly increased, whereas the level of IGFBP-4 expression was not altered. In the spleen, IGFBP-6 expression was not altered by IGF-II overexpression, whereas IGFBP-3 expression was strongly increased. The differences in IGFBP expression, and the difference in response of these IGFBPs to IGF-II overexpression in thymus and spleen suggests an important role of these proteins in growth regulation of both lymphoid tissues. We speculate that an increase of IGFBP-3 expression together with changes in expression of other IGFBPs, inhibits IGF-II stimulated growth in the spleen by an autocrine-/paracrine pathway.

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We adapted the Biolistics Particle Delivery System for the introduction of DNA into Xenopus embryos, allowing us to modulate the expression of different genes at specific time points during development. In the present study we applied the Biolistics method to the study of the wnt-engrailed signaling cascade in the developing Xenopus embryo. We show that ectopic expression of Xwnt-1 and Xwnt-5C is sufficient to activate specifically XEn-1 and not XEn-2.

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Expression of the Engrailed-1 (XEn-1) gene was studied in Xenopus embryogenesis by Northern blot analysis and whole-mount in situ hybridization. One transcript of 2.2 kb was detected from stage 17 (midneurula) onwards, until stage 47 (swimming tadpole). The expression pattern of the XEn-1 gene as revealed by in situ hybridization can be divided in three regions. The first domain of transient expression appears at the midneurula stage (st. 17) in the anterior part of the neural fold, forming a complete ring of positive cells at the mid/hindbrain border after neural tube closure. A second region of transient expression is detected as groups of ventro-lateral cells in the spinal cord and the hindbrain from late-neurula till tadpole stages. A third area of transient expression of XEn-1 is formed by the anterior part of the developing pronephros. Comparison of XEn-1 expression at the mid/hindbrain border with that of the Xenopus wnt-1 and engrailed-2 genes reveals that XEn-1 and Xwnt-1, in contrast to XEn-2, are both detected in a narrow stripe of positive cells in this region. Analysis in exogastrulated embryos reveals that expression of XEn-1 and Xwnt-1, but not XEn-2, is induced by planar signaling in the presumptive midbrain. Of the three genes only XEn-1 is expressed in the floorplate at the mid/hindbrain border, while Xwnt-1 is expressed in adjacent cells in the neural ectoderm. The results suggest that in vertebrates at the interface between cells in the floorplate and in the paraxial neuroectoderm, at the limited region of the mid/hindbrain border, En-1 interacts with wnt-1 in a signaling pathway analogous to the engrailed/wingless signaling in the parasegments of the Drosophila embryo.

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The Xwnt-5C gene is expressed in Xenopus embryos from the early gastrula stage onwards. The transcription of Xwnt-5C is regulated differentially with respect to transcript size, timing and localization. To gain insight into the generation of the Xwnt-5C expression pattern, we started to analyze the transcriptional regulation of this gene. We isolated Xwnt-5C genomic DNA sequences. By microinjection of chimaeric reporter constructs into Xenopus embryos we demonstrate that the upstream region contains a promoter functional in vivo. Of the several putative binding sites for trans-acting factors, present in a minimal promoter fragment, some have been studied in more detail. Mutations in an octamer motif and in an AP-2 consensus sequence interfere with the activity of the Xwnt-5C minimal promoter. In vitro binding assays with extracts from gastrula stage Xenopus embryos show that the octamer motif of the Xwnt-5C promoter can bind several Octamer binding factors, one of which is Oct1.

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We have studied the spatial expression of the Xenopus GR in early embryos by whole-mount in situ hybridization. At the gastrula stage, GR mRNA is localized in the dorsal ectoderm. By the early neurula stage, GR transcripts were detected along the notoplate. Between mid and late neurula stages, GR mRNA was not detectable. At the tailbud stage, GR mRNA was found in the anterior part of the embryo, including the cement gland, eyes, brain, the foregut, stomodeal-hypophyseal anlage, the olfactory placodes, head mesenchyme and somites. Injection of Xenopus GR RNA into zygotes followed by treatment dexamethasone from the blastula stage onwards inhibits early differentiation. Expression of Xbra, gsc and histone H3 genes in these embryos is not inhibited, indicating that the GR effects are not due to a general squelching effect on transcription.

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The Xenopus homolog of the proto-oncogene wnt-1 (int-1) is transiently expressed during neurula and tailbud stages of early development. To determine the mechanisms involved in the transcriptional regulation of the Xwnt-1 gene, we isolated Xwnt-1 genomic sequences. The promoter activity of the 5' flanking region of the gene was analysed by microinjection of chimeric luciferase reporter constructs into embryos. It is shown that the proximal 220 bp of the Xwnt-1 promoter is able to confer activation of the reporter gene at the early neurula stage. DNaseI footprinting analysis of the proximal promoter region with nuclear protein extracts from neurula stage embryos revealed five protein binding regions. Deletion and mutation analysis shows that one of these five protein binding regions is essential for promoter activity. Gel shift experiments indicate that a sequence element resembling the GT-I and GT-II motifs of the SV40 enhancer is important for the Xwnt-1 promoter activity in vivo.

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Myositis is a rare manifestation of Lyme disease of unknown pathogenesis. This study describes the course of disease in eight patients with Lyme disease, aged 37-70 years, all of whom were suffering from histologically proven myositis. The clinical, electrophysiological, and myopathological findings are reported. One patient showed signs and symptoms of myositis of all limbs. In six patients myositis was localized in the vicinity of skin lesions, arthritis or neuropathy caused by Borrelia burgdorferi. In another patient suffering from pronounced muscle weakness of the legs and cardiac arrest, inflammation of the myocardium, the conducting system and skeletal muscles was revealed at autopsy. Muscle biopsy revealed lymphoplasmocellular infiltrates combined with few fibre degenerations in three patients. The lymphoplasmocellular infiltrates were found predominantly in the vicinity of small vessels. Several spirochetes were stained in six of seven muscle biopsy samples by means of the immunogold-silver technique. Culturing of B. Burgdorferi from the muscle biopsy samples was, however, unsuccessful. Antibiotic treatment succeeded in curing the myositis in four of six patients. In one patients signs and symptoms improved. One patient died from cardiac arrest caused by myocarditis and Guillain-Barré syndrome. The outcome is unknown in one patient. Clinical and myopathological findings indicate that Lyme myositis can be caused either by local spreading of B. burgdorferi or an unknown antigen or toxin from adjacent tissues or haematogenously.

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Using cloned histone gene variants, the expression of three types of histone gene clusters was studied at the RNA level during early development in Xenopus laevis. For each histone class the number of mRNA molecules per embryo strongly decreased upon ovulation and steadily increased during early development, with a slight decrease at the neurula stage. Variation of the stringency of hybridization revealed that none of the histone genes probed is specifically and uniquely expressed at any time point in embryogenesis. The observed variation of histone mRNA content with time after fertilization is consistent with what is known about rate constants for RNA synthesis and degradation and about histone mRNA storage in the oocyte, provided that approximately 11 h after fertilization a regulatory transition is proposed (KOSTER, DESTREE and WESTERHOFF (1988) J. Theor. Biol. 135, 139-167). Similarly, the observed amounts of histone mRNA could well be sufficient to direct the synthesis of the required amount of histone protein. Control Analysis reveals that late in development, the histone to DNA ratio is controlled virtually equally strongly by the rate constants of DNA replication, transcription and translation. The control coefficient for RNA degradation is only a little smaller, whereas that for proteolysis is negligible. Indeed, the deceleration of DNA synthesis around the Mid Blastula Transition (some 8 h after fertilization in our studies) is a regulatory step that is essential in order to allow Xenopus to synthesize sufficient histones to structure its DNA; otherwise the embryo would run out of histone protein and histone mRNA soon thereafter. A model that assumes that the lengthening of the cell cycle around the Mid Blastula Transition it itself a response to the decrease in the concentration of histone protein not complexed to DNA, is shown to account for the dynamics of histone and DNA synthesis during the first 50 h after fertilization.

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Using literature data for transcriptional and translational rate constants, gene copy numbers, DNA concentrations, and stability constants, we have calculated the expected concentrations of histones and histone mRNA during embryogenesis of Xenopus laevis. The results led us to conclude that: (i) for X. laevis the gene copy number of the histone genes is too low to ensure the synthesis of sufficient histones during very early development, inheritance from the oocyte of either histone protein or histone mRNA (but not necessarily both) is necessary; (ii) from the known storage of histones in the oocyte and the rates of histone synthesis determined by Adamson & Woodland (1977), there would be sufficient histones to structure the newly synthesized DNA up to gastrulation but not thereafter (these empirical rates of histone synthesis may be underestimates); (iii) on the other hand, the amount of H3 mRNA recently observed during early embryogenesis (Koster, 1987, Koster et al., 1988) could direct a higher and sufficient synthesis of H3 protein, also after gastrulation. We present a quantitative model that accounts both for the observed H3 mRNA concentration as a function of time during embryogenesis and for the synthesis of sufficient histones to structure the DNA throughout early embryogenesis. The model suggests that X. laevis exhibits a major (i.e. some 14-fold) reduction in transcription of histone genes approximately 11 hours after fertilization. This reduction could be due to a decrease in the number of transcribed histone genes, a decreased rate constant of transcription with continued transcription of all the histone genes, and/or a reduction in the time during the cell cycle in which histone mRNA synthesis takes place. Alternatively, the histone mRNA stability might decrease approximately 16-fold 11 hours after fertilization.

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In eukaryotes pre-tRNA species are processed at the 5' end by an endonuclease. Here we describe the first characterization of the structure of a eukaryotic pre-tRNA 5' processing endonuclease. The 5' pre-tRNAase, isolated from X. laevis ovaries, copurifies with a 16S macromolecular complex consisting of at least 14 polypeptides ranging in MW from about 20,000 to 32,000. These polypeptides comprise a cylindrical particle, apparently organized as a stack of four rings, similar or identical to a ubiquitous eukaryotic subcellular particle described in the literature over the past 15 years. Similar copurification is observed for the enzyme from HeLa cells, suggesting that the X. laevis enzyme is representative of a general class of eukaryotic pre-tRNA 5' processing nuclease.

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Using previously cloned Xenopus nucleosomal core histone genes as hybridization probes, a genomic DNA library of Xenopus laevis was screened for histone gene clusters. From over 200 histone-gene containing clones identified, 36 were selected as possibly containing H1 histone genes by hybridization to a probe derived from a sea urchin H1 histone gene. These 36 clones were further analyzed by hybrid-selected translation for the definitive presence of H1 histone genes. The genes for three different H1 histone variants were found: H1A , H1B and H1C . Mapping of the histone genes within each clone showed that at least three different gene arrangements can occur within a cluster and that the type of H1 histone variant present in a cluster may be related to the cluster type. S1-mapping experiments indicated that histone genes found in different cluster-types can be expressed in oocytes. Also, the H1 gene found in one cluster-type was expressed in at least three different cell-types: oocytes, gastrula-stage embryos, and erythroblasts.

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