Identification of a novel mutation of the EDA gene in X-linked hypohidrotic ectodermal dysplasia.

This study aimed to identify the disease-causing mutation in the ectodysplasin A (EDA) gene in a Chinese family affected by X-linked hypohidrotic ectodermal dysplasia (XLHED). A family clinically diagnosed with XLHED was investigated. For mutation analysis, the coding region of EDA of 2 patients and 7 unaffected members of the family was sequenced. The detected mutation in EDA was investigated in 120 normal controls. A missense mutation (c.878T>G) in EDA was detected in 2 patients and 3 female carriers, but not in 4 unaffected members of the family. The mutation was not found in the 120 healthy controls and has not been reported previously. Our findings indicate that a novel mutation (c.878T>G) of EDA is associated with XLHED and adds to the repertoire of EDA mutations.

Synthesis and structure of a new scorpionate-dithio carboxyl oxovanadium complex and an organic dithio carboxyl compound.

A new complex of oxovanadium(IV), V(2)O(2)[(HB(pz)(3))(2)(pyrro)(2) (1) and a dimer-dithio carboxyl compound (C(5)H(8)NS(2))(2) (2) have been synthesized by the reaction of VOSO(4).nH(2)O with NaHB(pz)(3) and pyrrolidine dithio carboxylic acid ammonium salt. They were characterized by element analysis, IR spectra, UV-vis spectra and X-ray diffraction. Structural analyses of 1 and 2 gave the following parameters: 1, triclinic, P-1, a=7.732(4)A, b=14.285(8)A, c=17.802(9)A, alpha=101.314(8) degrees , beta=92.682(9) degrees , gamma=92.228(9) degrees , V=1923.6(18)A(3), and Z=4; 2, monoclinic, C2/c, a=13.857(2)A, b=10.4213(18)A, c=9.436(2)A, beta=97.099(2), V=1352.1(4)A(3), and Z=4. In complex 1, vanadium atom adopts a distorted tetragonal bipyramid structure, which is typical for oxovanadium(IV) complexes. Compound 2 is a dimer-dithio carboxyl compound with S-S bond. In addition, thermal analysis was performed for analyzing the stabilization of the complexes.

The first organo-templated cobalt phosphate with a zeolite topology.

An organic molecule containing cobalt phosphate (denoted CoPO-GIS) which is isostructural with the zeolite gismondine has been synthesized under solvothermal conditions by using [Co(en)3]Cl3 and phosphoric acid as the reactants and ethylene glycol as the solvent. CoPO-GIS ((H3NCH2CH2NH3)0.5.CoPO4) crystallizes in the monoclinic space group C2/c with a = 14.744(3) A, b = 8.850(3) A, c = 10.062(3) A, beta = 131.609(19) degrees, and Z = 8. The structure consists of a 3-D network of strictly alternating CoO4 and PO4 tetrahedra interconnected by oxygen bridges, and the charge-balancing diprotonated ethylenediamine cations are highly ordered in the cages of the CoPO4 framework. CoPO-GIS is the only amine-containing cobalt phosphate with a known zeolite topology.

Insertion of tandem direct repeats consisting of avian leukosis virus LTR sequences into the inverted repeat region of Marek's disease virus type 1 DNA.

The BC-1 strain DNA of Marek's disease virus type 1 (MDV1) at high-passage in culture was found to contain tandem direct repeats of the complete long terminal repeat (LTR) sequence of avian leukosis virus RAV0 strain as the repeat unit within the short inverted repeats of the MDV1 DNA. Since the attenuated BC-1 strain grows well in cultured cells, the insertion site for retroviral DNA sequence within the short inverted repeat of MDV1 DNA is not essential for viral growth in culture.

Asymmetric deletion of the junction between the short unique region and the inverted repeat does not affect viral growth in culture and vaccine-induced immunity against Marek's disease.

To construct an effective recombinant Marek's disease virus type 1 (MDV1), we localized a stable insertion site for expression of the Escherichia coli lacZ gene near or within the short inverted repeats of MDV1 strain K554 DNA. A stable recombinant MDV1 was obtained by deleting the junction region between the short unique sequence (Us) and the internal short inverted repeat (IRs). The recombinant MDV1 replicated in cultured cells as well as the parental viral DNA. Antibodies against both MDV1 antigen and beta-galactosidase encoded by the lacZ gene were detected in the sera of chickens immunized with the virus, and persisted for at least 16 weeks. Moreover, the recombinant virus conferred protection upon chickens against a challenge with virulent MDV1. These results demonstrated that the Us-IRs junction region is an effective site for the insertion of foreign genes from which to construct a polyvalent live vaccine for poultry. Analysis of the Us-IRs junction region which was deleted from the parental MDV1 indicated that there is a tandem direct repeat of a 220-bp exists within the short internal and terminal inverted repeats of avirulent MDV1 K554 strain DNA. The 220-bp sequence was well conserved among DNAs from various strains. The number of the repeat units may differ between the IRs and TRs or among various MDV1 strain DNAs.

Marek's disease virus type 1-specific phosphorylated proteins pp38 and pp24 with common amino acid termini are encoded from the opposite junction regions between the long unique and inverted repeat sequences of viral genome.

The nucleotide sequence of the junction region between the long unique (UL) and terminal inverted repeat (TRL) sequences of Marek's disease (MD) virus type 1 (MDV1) DNA revealed the presence of a rightward open reading frame of 155 amino acids. The ORF inserted into an eukaryotic expression vector transiently expressed an antigen in the cytoplasm of COS7 cells which reacted with the monoclonal antibody M21 against an MDV1-specific phosphorylated protein complex consisting of at least the proteins pp38 and pp24. In addition, RNA synthesized in vitro from the ORF under the control of the T7 promoter was translated in vitro using rabbit reticulocyte lysates. A polypeptide of about 24 kDa was immunoprecipitated with M21 antibody. Thus, the MDV1-specific phosphorylated proteins pp38 and pp24 with common amino termini are encoded in the opposite junction regions between the UL and IRL and between the UL and TRL, respectively, of the MDV1 genome. The pp38 gene is transcribed leftward from the viral genome, while the pp24 gene was shown here to be transcribed rightward in a MD tumor cell line as well as in cells productively infected with MDV1.

Expression of a novel Marek's disease virus type 1 (MDV1)-specific protein p40 in insect cells by a baculovirus vector and in chick embryo fibroblasts infected with MDV1.

We found that the open reading frame of 1053 nucleotides within the short unique sequence of Marek's disease virus (MDV) type 1 (MDV1) expresses a 40 kDa protein, using a baculovirus vector in insect cells. Mouse antisera against the 40 kDa protein reacted with a 40 kDa protein in MDV1-infected cells. A cross-reactive polypeptide of 37 kDa was also detected in cells infected with herpesvirus of turkey.

Marek's disease virus protein kinase gene identified within the short unique region of the viral genome is not essential for viral replication in cell culture and vaccine-induced immunity in chickens.

The open reading frame (ORF) of 1206 bp within the short unique region (Us) of Marek's disease virus type 1 (MDV1) shows significant homology with the herpes simplex virus type 1 US3 gene encoding protein kinase (PK). The lacZ gene of Escherichia coli was inserted within the ORF, designated MDV1-US3, of MDV1 K544 strain DNA by homologous recombination. The plaque-purified recombinant MDV1 stably expressed the beta-galactosidase encoded by the inserted lacZ gene in infected cells and replicated well as the parental K544 strain. Antibodies against both MDV1 antigen and beta-galactosidase were detected in the sera of chickens immunized with recombinant MDV1. Chickens vaccinated with the recombinant MDV1 were protected from challenge with virulent MDV1. The MDV1 US3 gene expressed by a baculovirus vector encoded a 44-kDa protein. Mouse antisera against the 44-kDa protein reacted with two proteins of 44 and 45 kDa in extracts of cells infected with MDV1 but not with MDV types 2 or 3. The PK activity was detected in immune complexes of the anti-44-kDa sera with extracts of cells infected with MDV1 but not with the recombinant MDV1. Thus, PK encoded from the MDV1-US3 is not essential for virus replication in cell culture and vaccine-induced immunity.

Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 by using polymerase chain reaction DNA amplification.

Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 (MDV1) was attempted by polymerase chain reaction (PCR) using the primers chosen from the sequence within the long inverted repeats of MDV1 DNA. PCR of the DNAs extracted from oncogenic-strain-infected cells and Marek's disease tumor cell lines produced a major product containing two or three copies of 132-base-pair (bp) repeat units, whereas PCRs of the DNAs extracted from nononcogenic-strain-infected cells yielded amplified products with various sizes corresponding to the number of 132-bp repeat units. The primers chosen from the glycoprotein A genes of MDV1 and herpesvirus of turkeys also were used for determination of their serotype specificity. The PCR procedure was found to be a simple and sensitive procedure for identification of MDV1 and herpesvirus of turkeys and for estimation of oncogenicity of MDV1.