Detection of the Nicotiana rustica chloroplast genome coding for the large subunit of Fraction I protein in a somatic hybrid in which only the N. tabacum chloroplast genome appeared to have been expressed

Nine plants were produced from anthers of a somatic hybrid which had been obtained by fusion of Nicotiana tabacum L. and N. rustica L. protoplants. As determined by electrofocusing, the Fraction I protein of the original somatic hybrid had largesubunit polypeptides exclusively of the N. tabacum type. Two of the plants regenerated from anthers contained Fraction-I-protein large subunits exclusively of the N. rustica type. Since each plant was regenerated from a single cell, the somatic hybrid must have had cells containing both the N. tabacum and N. rustica chloroplast genome although the latter was not expressed. Possibilities to account for this non-expression of a chloroplast genome in the somatic hybrid are discussed.     

High mobility group nonhistone chromosomal proteins also exist in Tetrahymena.

High mobility group (HMG) nonhistone chromosomal proteins have been shown to exist also in the ciliated protozoan Tetrahymena pyriformis. One or two histone-like components were extracted with 0.25 M HCl from the chromatin, in addition to five histone species. These proteins were also extracted selectively with 0.5 M HClO4, 0.35 M NaCl, or 4 mM spermidine, together with H1 histone, and were characterized as HMG proteins on the basis of the following criteria: high mobilities on polyacrylamide gel electrophoresis, relatively low molecular weights, amino acid compositions rich in lysine and glutamic acid, and relative contents in chromatin. This extends the distribution of the HMG proteins to all four eukaryotic kingdoms, and suggests the possibility that they have some universal role in chromatin structure and function.     

Identification of suberimidate cross-linking sites of four histone sequences in H1-depleted chromatin. Histone arrangement in nucleosome core

The arrangement of 8 histones in the nucleosome core has been investigated by identifying the sites of 4 histone sequences cross-linked with a bifunctional amino-group reagent, dimethyl suberimidate, selected from among 4 diimidoesters of various linker lengths examined. H1-depleted calf thymus chromatin was allowed to react with 14C-labeled suberimidate at pH 8.5 and 0 degrees C. The cross-linked chromatin was then digested exhaustively with trypsin. Almost all the histone fragments were released from the chromatin with 0.25 M HCl and chromatographed on several columns and on paper. Cross-linked peptides were detected by analyzing the content of radioactive suberimidoylbislysine after acid hydrolysis. The chromatographic procedure developed here showed that the whole histone fragments contained 29 mol% of the total linked reagent as suberimidoylbisylsine. The 5 finally purified cross-linked peptides were identified from the total and N-terminal amino acids of each pair of peptides separated by two-dimensional cellulose thin layer chromatography after cutting the linker by ammonolysis. Thus, intramolecular cross-linking was found between Lys-5 and Lys-9 of H2A, and Lys-34 and Lys-85 of H2B, while intermolecular cross-linking was found between Lys-24 (or 27) of H2B and Lys-74 of H2A, Lys-85 of H2B and Lys-91 of H4, and Lys-120 of H2B and Lys-115 of H3 and/or Lys-77 of H4. Most of these lysine residues are located in the DNA-binding segments of the 4 histone sequences identified previously [Kato, Y. & Iwai, K, (1977) J. Biochem. 81, 621--630]. All the 5 or 6 cross-links can be located in a heterotypic tetramer consisting of one molecule each of H2A, H2B, H3, and H4, and a model of the histone arrangement in the tetramer is proposed. Two such tetramers may compose to the histone octamer in the nucleosome core.     

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. d-Tyrosine was used for the control. α-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.     

Studies on the sites in histones phosphorylated by adenosine 3':5'-monophosphate-dependent and guanosine 3':5'-monophosphate-dependent protein kinases.

Adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) purified from silkworm pupae phosphorylated five major fractions of calf thymus histone, whereas guanosine 3':5'-monophosphate-dependent protein kinase (protein kinase G) purified from the same organism reacted preferentially with H1, H2A, and H2B histones. Amino acid analysis of the phosphopeptides which were obtained by proteolytic digestion revealed that both protein kinases A and G showed the abilities to phosphorylate the same serine hydroxyl groups in H1 and H2B histones. Both protein kinases reacted with Ser-38 in H1 histone. With H2B histone as substrate protein kinase A phosphorylated Ser-32 as well as Ser-36, whereas protein kinase G reacted preferentially with Ser-32 and the reaction with Ser-36 was very slow. H3 and H4 histones were practically inactive substrates for protein kinase G. Although H2A histone has not been analyzed, the evidence has raised a possibility that protein kinase G utilizes a portion of the substrate proteins for protein kinase A.     

Isolation of cDNA Clones for Epidermis-Specific Genes of the Ascidian Embryo

The present investigation was conducted to isolate cDNA clones that correspond to epidermis-specific genes of the ascidian embryo. When cleavage of fertilized eggs of Halocynthia roretzi is blocked by treatment with cytochalasin B and the arrested eggs are reared as one-celled embryos for about 30 hr, they develop features of differentiation of the epidermis only. Translation in vitro of poly(A)⁺ RNA from cleavage-arrested embryos and analysis of the products by two-dimensional gel electrophoresis revealed several predominant polypeptides that were not detected in a similar analysis of fertilized eggs, suggesting the appearance of epidermis-specific mRNAs in cleavage-arrested embryos. A cDNA library was constructed from arrested one-celled embryos. Differential screening of the library with a total cDNA probe from cleavage-arrested embryos and with a similar probe from fertilized eggs yielded eight different cDNA clones specific for the cleavage-arrested embryos. Northern blot analysis revealed that the mRNAs that corresponded to these cDNAs were present in normal tailbud embryos. In addition, in situ hybridization of whole-mount specimens showed that the mRNAs were restricted to the epidermal cells of tailbud embryos.     

Targeted gene modification in mouse ES cells using integrase‐defective lentiviral vectors

Lentiviral vectors efficiently integrate into the host genome of both dividing and nondividing cells, and so they have been used for stable transgene expression in biological and biomedical studies. However, recent studies have highlighted the risk of insertional mutagenesis and subsequent oncogenesis. Here, we used an integrase‐defective lentiviral (IDLV) vector to decrease the chance of random integration and examined the feasibility of lentiviral vector‐mediated gene targeting into murine embryonic stem (ES) cells. After transduction with wild‐type lentiviral vectors, none of the 512 G418 resistant clones were found to be homologous recombinant clones. Although the transduction efficiency was lower with the IDLV vectors (5.9% of wild‐type), successful homologous recombination was observed in nine out of the 941 G418 resistant clones (0.83 ± 1.32%). Pluripotency of the homologous recombinant ES cells was confirmed by the production of chimeric mice and subsequent germ line transmission. Because lentiviral vectors can efficiently transduce a variety of stem cell types, our strategy has potential relevance for secure gene‐manipulation in therapeutic applications. genesis 47:217–223, 2009. © 2009 Wiley‐Liss, Inc.