Keiichi Hosokawa

Identification of a Human Type II Receptor for Bone Morphogenetic Protein-4 That Forms Differential Heteromeric Complexes with Bone Morphogenetic Protein Type I Receptors

Bone morphogenetic proteins (BMPs) comprise the largest subfamily of TGF-β-related ligands and are known to bind to type I and type II receptor serine/threonine kinases. Although several mammalian BMP type I receptors have been identified, the mammalian BMP type II receptors have remained elusive. We have isolated a cDNA encoding a novel transmembrane serine/threonine kinase from human skin fibroblasts which we demonstrate here to be a type II receptor that binds BMP-4. This receptor (BRK-3) is distantly related to other known type II receptors and is distinguished from them by an extremely long carboxyl-terminal sequence following the intracellular kinase domain. The BRK-3 gene is widely expressed in a variety of adult tissues. When expressed alone in COS cells, BRK-3 specifically binds BMP-4, but cross-linking of BMP-4 to BRK-3 is undetectable in the absence of either the BRK-1 or BRK-2 BMP type I receptors. Cotransfection of BRK-2 with BRK-3 greatly enhanced affinity labeling of BMP-4 to the type I receptor, in contrast to the affinity labeling pattern observed with the BRK-1 + BRK-3 heteromeric complex. Furthermore, a subpopulation of super-high affinity binding sites is formed in COS cells upon cotransfection only of BRK-2 + BRK-3, suggesting that the different heteromeric BMP receptor complexes have different signaling potential.

Expression of exogenously introduced bacterial chloramphenicol acetyltransferase genes in Xenopus laevis embryos before the midblastula transition

SummaryPrevious papers have reported that DNAs exogenously injected into Xenopus laevis fertilized eggs are expressed only at and after the midblastula transition (MBT). We have injected fertilized eggs of Xenopus laevis with circular plasmids that contained bacterial chloramphenicol acetyltransferase (CAT) genes connected to the promoter of viral genes (pSV2CAT and pAd12.E1aCAT) or the Xenopus cardiac actin gene (actin-CAT fusion gene), and examined whether these DNAs are expressed during the stage before the MBT. We found that expression of CAT enzyme can be detected before the MBT when CAT genes connected to viral promoters were injected. The activity was low during the cleavage stage on a per-embryo basis; however, the time course of the accumulation of the CAT enzyme activity roughly paralleled the increase in cell number. Therefore, CAT enzyme activity per cell was constant during cleavage and did not change dramatically before and after the MBT stage. CAT mRNA level, detected by CAT antisense RNA, was roughly proportional to the levels of CAT enzyme. Therefore, we assume that the observed enzyme activity reflects the transcriptional activity of injected CAT genes before and after the MBT. When the actin-CAT fusion gene was injected, however, no enzyme activity was detected until embryos had reached the neurula stage, a stage when endogenous actin genes are first activated. On the basis of these results, we conclude that the concept of an initial transcriptional activation of exogenous genes at amphibian MBT has to be changed. We propose that the expression of polymerase-II-transcribed genes is regulated by the nature of the promoters connected to the genes rather than by changes associated with MBT.

Characterization of the constituent polypeptides of the extracellular hemoglobin from Lumbricus terrestris: heterogeneity and discovery of a new linker chain L4.

The extracellular hemoglobin of Lumbricus terrestris comprises four oxygen binding chains, a, b, c, d, and three linker chains L1, L2, L3 as major components. A stoichiometry of the whole molecule has been proposed on the basis of these chains, with a total number of 216 chains: forty-eight chains of each oxygen binding chain and eight molecules of each linker chain. We have isolated additional minor components by HPLC and two-dimensional gel electrophoresis. The following biochemical characterizations have been made. (i) All components so far reported, the heme-containing chains a, b, c, d, and linker chains L1, L2, L3 and a new minor polypeptide, L4, were mapped on a two-dimensional gel. Their estimated isoelectric points were between 4.7 and 5.9. (ii) The sequences of several peptides including the unique N-terminal peptide from linker L4 show that it can be considered as a duplicated gene product with a similar mass. (iii) Chain d2 was isolated and found to correspond to the minor chain previously pointed out by Shishikura et al. (J. Biol. Chem. 262 (1987) 3123-3131). (iv) The major chain d1 has serine at position 7 from the N-terminus. This is not consistent with previously reported glycine (Shishikura et al., J. Biol. Chem. 262 (1987) 3123-3131).

Invitro transcription of a chromatin-like complex of major core protein VII and DNA of adenovirus serotype 2

Major core protein VII of adenovirus serotype 2 (Ad2) is thought to play a role as a histone octamer in eukaryotic cells. We compared the template activity of the VII-DNA complex formed in vitro with that of protein-free DNA. Hybridization assay of in vitro transcripts showed that transcription from regions located in the middle of Ad2 DNA decreased when Ad2 DNA formed a complex with VII. This suggests that the chromatin structure plays a role in regulation of transcription of the adenovirus genome.

The structure of adenovirion chromatin revealed by ultraviolet light-induced cross-linking

Abstract Upon irradiating adenovirion by ultraviolet light, viral DNA and a part of viral protein cosedimented faster than free DNA when examined in the presence of 4 M guanidine HC1 in sucrose gradient, indicating that protein became tightly bound to DNA. By sodium dodecylsulfate polyacrylamide gel electrophoresis, it was verified that the protein is cross-linked covalently to DNA and that polypeptide VII is the only polypeptide species present. Based on the available data on the size of nuclease digested fragments, a model of adenovirion chromatin consisting of a nucleosome-like subunit of polypeptide VII-hexamer was constructed.

ANTI-INTERFERON ACTIVITY OF ADENOVIRUS-2-ENCODED VAI AND VAII RNAS IN TRANSLATION IN CULTURED HUMAN CELLS

The mode of anti-interferon action of VAI and VAII RNAs of adenovirus type 2 (Ad2) was studied by transfecting interferon-alpha (IFN-alpha)-treated KB cells in culture with a plasmid construct containing the VAI or VAII RNA gene and an SV40 promoter-chloramphenicol acetyltransferase (CAT) gene construct as reporter (pSV2-CAT). The longer the treatment of KB cells with IFN-alpha (2,000 IU/ml) lasted, the higher was the inhibition of CAT expression. A maximum of 76% inhibition was attained without pronounced cytotoxicity during 48 h of treatment. The earlier the VAI RNA gene was transfected, the higher was the enhancement of CAT expression. CAT activity increased from 113 to 157% in normal cells and 200-400% in IFN-alpha treated cells, as compared with the corresponding controls without VAI RNA transfection. The level of CAT mRNA was neither appreciably decreased by IFN-alpha treatment, nor detectably increased by VAI or VAII RNA. The effect of VA RNA thus appeared to be on translation rather than on transcription. The relative constancy of the level of CAT mRNA indicated that IFN-alpha inhibition of CAT expression was not due to the activation of RNase L, but due mainly to translational repression. The level of VAII RNA expressed was only 9-13% of that of VAI RNA. Nevertheless, VAII RNA gene transfection stimulated CAT activity to 112% of the control in non-IFN-alpha-treated cells, and 126-182% in IFN-alpha-inhibited cells. When IFN-alpha treatment was started late after VAI RNA cotransfection, CAT expression increased to 169% which was higher than the expression in cotransfected control cells without IFN-alpha treatment. The enhanced level of CAT activity was in remarkable contrast to the IFN-alpha inhibited level of 25% without VA RNA co-transfection when IFN-alpha was added upon seeding. The enhanced CAT activity in cells treated late with IFN-alpha could be ascribed to higher levels of VA RNAs.

DNA methylation in the promoter of ribosomal RNA genes in human cells as determined by genomic sequencing

Many RNA polymerase II‐ or III‐transcribed genes are inactive when their promoter is methylated at critical CpG dinucleotides. We have applied the genomic sequencing method and a direct DNA blotting technique to analyze the extent of DNA methylation in the 5′‐CpG‐3′ rich promoter region of the RNA polymerase I‐transcribed ribosomal RNA genes (rDNA) in DNA from primary human cells, primary human tumor cells and human cell lines. In none of the analyzed primary human cells and primary human tumor cells was the DNA in the rDNA promoter region found to be detectably methylated. In contrast, in some of the cell lines this promoter is methylated in all 5′‐CpG‐3′ dinucleotides in the majority of the approximately 200 ribosomal RNA gene copies. In actively growing cells, rDNA gene activity is a prerequisite for cell viability. The high levels of DNA methylation in the promoter region of rDNA in the human cell lines raise questions on the role of promoter methylation in these RNA polymerase I‐transcribed genes. It is, however, conceivable that a subset of the about 200 rDNA copies per haploid genome have escaped methylation and account for the rRNA synthesis in these cell lines. Alternatively, complete 5′‐CpG‐3′ promoter methylation may be compatible with promoter activity as demonstrated for certain viral genomes.

Temporally uncontrolled expression of linearized plasmid DNA which carries bacterial chloramphenicol acetyltransferase gene withXenopus cardiacα-actin promoter after injection intoXenopus fertilized eggs

SummaryCircular and linearized plasmid DNA which contained bacterial chloramphenicol acetyltransferase (CAT) gene connected toXenopus cardiacα-actin promoter was injected intoXenopus fertilized eggs to study their expression in the course of early embryonic development. While circular DNA was slightly replicated and expressed only after embryos reached neurula stage, linearized DNA formed a large amount of concatemers, and was expressed as early as at blastula stage, or about 14 hr earlier than the time of circular DNA expression. Similarly earlier expression of linearized DNA occurred slightly more strongly when the DNA was injected into presumptive dorsal than in ventral blastomeres at 4-cell stage, and the expression was not affected when embryos were dissociated at blastula stage and their cells were cultured under reaggregating or nonreaggregating conditions. These results show that although circular actin-CAT fusion gene is expressed during development according to endogenous temporal control, the expression of linearized DNA deviates from such developmental control even though it contains intact promoter ofα-actin gene. It is then recommended that study of the control of the expression of exogenously-introduced DNA inXenopus fertilized eggs should be carried out with circular but not linearized plasmids.

Effects of thiocyanate on cytosol androgen receptor from Shionogi carcinoma 115

The effects of KSCN and other chaotropic salts on the androgen receptor in cytosol of Shionogi carcinoma 115 were studied by means of charcoal adsorption assay and sucrose gradient centrifugation. When KSCN or NaSCN was added to the [3H]-dihydrotestosterone-cytosol mixture at the final concentration of 0.5 M, the androgen binding to the cytosol receptor was considerably inhibited. The inhibition reached maximum within 5 h at 0 degrees C and was dependent on the kind of chaotropic anion added: the potency of inhibitory effect in descending order was KSCN greater than KI greater than KBr greater than KCl. The inhibition was not observed in the estradiol-receptor interaction with KSCN or NaSCN up to 0.5 M. When 0.5 M KSCN-treated androgen-cytosol mixture was subjected to gel filtration or (NH4)2SO4 fractionation to remove the salt, a partial recovery (30-40%) in specific binding activity was observed. The binding activity of androgen receptor was unaffected by a treatment with KSCN up to 0.1 M and the androgen-receptor complex sedimented in a 5S form in 0.1-0.3 M KSCN, 0.5 M KCl or 0.5 M KBr. These results suggest that the binding activity of androgen receptor is more susceptible than that of estrogen receptor to chaotropic salts which cause impairment in intramolecular hydrophobic interactions.

Salt extractability of nuclear receptor-estrogen complex from the rat uterus

Estrogen receptors in the nuclei of rat uterus were determined 1 or 6 h after the injection of [3H]estradiol. Ratio of 0.4 M KCl-extractable to -resistant receptor-estradiol complex was constant during this time interval. Three consecutive extractions took out about 95% of receptor-estradiol complex either 1 or 6 h after injection. Extraction with KCl before the exchange assay reduced the amount of salt-resistant nuclear receptor-estradiol complex. However, when exchange incubation with [3H] estradiol was done before KCl extraction, salt-resistant receptor-estradiol complex was significantly increased, even after three consecutive extractions.