Integration of exogenous DNA into mouse embryonic stem cell chromosomes shows preference into genes and frequent modification at junctions.

Chromosomal integration of exogenous DNA in mammalian cells allows stable gene expression for a variety of biological applications. Although it is presumably mediated by DNA repair machinery, little is known regarding site preferences and other characteristics. We isolated and analyzed 256 chromosomal-plasmid DNA integration junctions from 158 plasmid integrants after electroporation in mouse embryonic stem (ES) cells.

Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

Background: Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR.

Methods: We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells.

The female-killing chromosome of the silkworm, Bombyx mori, was generated by translocation between the Z and W chromosomes.

Bombyx mori is a female-heterogametic organism (female, ZW; male, ZZ) that appears to have a putative feminizing gene (Fem) on the W chromosome. The paternally transmitted mutant W chromosome, Df(p ( Sa ) + ( p )W + ( od ))Fem, derived from the translocation-carrying W chromosome (p ( Sa ) + ( p )W + ( od )), is inert as femaleness determinant. Moreover, this Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome has been thought to have a female-killing factor because no female larvae having the Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome are produced.

Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors.

For gene therapy of inherited diseases, targeted integration/gene repair through homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to avoid potentially serious problems of random integration such as cellular transformation and gene silencing. Efficient sequence-specific modification of chromosomes by HR would also advance both biological studies and therapeutic applications of a variety of stem cells. Toward these goals, we developed an improved strategy of adenoviral vector (AdV)-mediated HR and examined its ability to correct an insertional mutation in the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse ES cells.

Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori.

In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W.

Detachment analysis of the translocated W chromosome shows that the female-specific randomly amplified polymorphic DNA (RAPD) marker, female-218, is derived from the second chromosome fragment region of the translocated W chromosome of the sex-limited p(B) silkworm (Bombyx mori ) strain.

The sex chromosomes of the silkworm, Bombyx mori, are designated ZW for the female and ZZ for the male. We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD) marker, designated Female-218, from the translocation-bearing W chromosomes. These W chromosomes contain a region of the second chromosome, which carries visible larval markers of the p loci.

Nested retrotransposons on the W chromosome of the wild silkworm Bombyx mandarina.

The W chromosome of the silkworms Bombyx mori or B. mandarina is recombinationally isolated from the Z chromosome and the autosomes. We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD), designated W-Yamato, derived from the W chromosome of the wild silkworm Bombyx mandarina.

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori.

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons.

Two novel Pao-like retrotransposons (Kamikaze and Yamato) from the silkworm species Bombyx mori and B. mandarina: common structural features of Pao-like elements.

To characterize the structural features common to Pao-like retrotransposons, we analyzed two lambda phage clones which contain the Pao-like elements from the silkworm species Bombyx mori and B. mandarinia, and copies of Pao itself and ninja of Drosophila simulans, amplified by PCR. We previously identified two randomly amplified polymorphic DNAs (RAPDs), W-Kamikaze and W-Yamato, from B.

A homologue of the Drosophila doublesex gene is transcribed into sex-specific mRNA isoforms in the silkworm, Bombyx mori.

The doublesex (dsx) gene is known as the final gene of the sex-determining cascade in Drosophila melanogaster. We have isolated a homologue of dsx in the silkworm, Bombyx mori, which has an epistatic feminizing gene located on the W chromosome. RT-PCR analysis indicated that B.

Molecular structure of a novel gypsy-Ty3-like retrotransposon (Kabuki) and nested retrotransposable elements on the W chromosome of the silkworm Bombyx mori.

We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD), designated W-Kabuki, derived from the W chromosome of the silkworm, Bombyx mori. To further analyze the W chromosome of B. mori, we obtained a lambda phage clone which contains the W-Kabuki RAPD sequence and sequenced the 18.

Identification and genetic mapping of RAPD markers linked to the densonucleosis refractoriness gene, nsd-2, in the silkworm, Bombyx mori.

In the silkworm, Bombyx mori, nonsusceptibility to B. mori densonucleosis virus type-2 (BmDNV-2) is controlled by a recessive gene, nsd-2 (nonsusceptibility to DNV-2). We investigated the genetic linkage between two random-amplified polymorphic DNA (RAPD) markers and the +nsd-2 gene.

A complete full-length non-LTR retrotransposon, BMC1, on the W chromosome of the silkworm, Bombyx mori.

In the silkworm, Bombyx mori, a non-long terminal repeat (non-LTR) retrotransposon, BMC1, is considered to be a LINE (long interspersed nuclear element)-like element. So far, a BMC1 containing two intact open reading frames (ORFs) has not been found. However, we discovered a complete full-length BMC1 on the W chromosome.

Molecular structure of the copia-like retrotransposable element Yokozuna on the W chromosome of the silkworm, Bombyx mori.

We discovered a novel retrotransposable element, designated Yokozuna, on the W chromosome of Bombyx mori. The size of this element is 4738 bp, including a 208-bp long terminal repeat (LTR) on one side and a 183-bp LTR on the other. This retrotransposable element is flanked by a 5-bp target site duplication, TAATT.

Identification of novel random amplified polymorphic DNAs (RAPDs) on the W chromosome of the domesticated silkworm, Bombyx mori, and the wild silkworm, B. mandarina, and their retrotransposable element-related nucleotide sequences.

Genomic DNAs were compared between males and females of the domesticated silkworm, Bombyx mori, strains C108, C137, J137, p50, and WILD-W (constructed by crossing a wild silkworm, B. mandarina, female with a male of strain C108) by polymerase chain reaction (PCR) with 700 arbitrary 10-mer primers. Four female-specific RAPDs (W-Kabuki, W-Samurai, W-Kamikaze, and W-Yamato) were found.

Genetic mapping of RAPD markers linked to the densonucleosis refractoriness gene, nsd-1, in the silkworm, Bombyx mori.

In the silkworm, Bombyx mori, nonsusceptibility to B. mori densonucleosis virus type-1 (BmDNV-1) is controlled by a recessive gene, nsd-1 (nonsusceptibility to DNV-1), located on the twenty-first chromosome. We investigated genetic linkage between five random amplified polymorphic DNA (RAPD) markers and the +nsd-1 gene.

[Pharmacokinetic and clinical studies of cefotiam during the perinatal period in pregnant women].

Pharmacokinetic and clinical studies of cefotiam (CTM) were carried out in pregnant women. The results obtained are summarized below. The concentration of CTM in amniotic fluid increased gradually up to 14.

[In vitro activity and clinical evaluation of aztreonam in the field of obstetrics and gynecology].

Antimicrobial activity of aztreonam (AZT) against 231 clinical isolates in the field of obstetrics and gynecology was determined by agar-plates dilution method. Almost of all strains of E. coli (108 strains) tested were susceptible to the concentration of 0.

[Fundamental and clinical studies of cefpiramide in the field of obstetrics and gynecology].

Fundamental and clinical studies of cefpiramide (CPM, SM-1652) a new semisynthetic cephalosporin were carried out in the field of obstetrics and gynecology. The results were obtained as follows: In vitro antibacterial activity of CPM against recent 255 clinical isolates was compared with those of cefazolin (CEZ), cefmetazole (CMZ) and cefoperazone (CPZ). CPM showed strong antibacterial activity against Staphylococcus, K.

Reduced DNA repair response of cells from carcinogen-induced rat liver altered foci exposed to chemical carcinogens in culture.

Cells were cultured from rat livers containing enzyme-altered foci induced by N-2-fluorenylacetamide and the level of DNA repair synthesis in response to several carcinogens was compared in gamma-glutamyl transpeptidase-positive foci cells and in enzyme-negative hepatocytes from the same livers. The level of unscheduled DNA synthesis elicited in foci cells by either the activation-dependent carcinogens N-2-fluorenylacetamide and diethylnitrosamine or the activation-independent carcinogens N-methyl-N'-nitro-N-nitrosoguanidine and 1-methyl-1-nitrosourea was significantly lower than that in hepatocytes. The results suggest that the repair of DNA damage by these altered cells is abnormal.

[Bacteriological study of cefminox in the field of obstetrics and gynecology].

Cefminox (CMNX, MT-141), a new cephem antibiotic, was determined of its antibacterial activity against 304 clinical isolates with following results. CMNX was inferior to CEZ or CMZ in the activity against 78 isolates of Staphylococcus sp., but it was superior to these antibiotics in the activity against 104 isolates of E.

Genotoxicity of a variety of mycotoxins in the hepatocyte primary culture/DNA repair test using rat and mouse hepatocytes.

Twenty-eight mycotoxins were studied in the hepatocyte primary culture/DNA repair test using rat and mouse hepatocytes. DNA repair synthesis was elicited by several compounds of unknown carcinogenicity, 5,6- dimethoxysterigmatocystin , versicolorins A and B, averufin , xanthomegnin , luteosporin , and chrysazin , as well as by the carcinogenic myocotoxins , aflatoxin B1, sterigmatocystin, luteoskyrin , ochratoxin A, azaserine, mitomycin C, and actinomycin D. The positive results with compounds of unknown carcinogenicity suggest that they are possibly genotoxic carcinogens.

Some toxic properties of a carcinogenic pyrrolizidine alkaloid, petasitenine.

Some toxic properties of petasitenine, a hepatocarcinogenic pyrrolizidine alkaloid, were confirmed in 3 parts of studies. Electron microscopic observation of rat liver on the stage of acute toxicity following administration of a toxic dose of petasitenine disclosed the distinctive changes i.e.

Glassy cell carcinoma of the uterine cervix.

Two cases of glassy cell carcinoma which is considered to be a poorly differentiated mixed adenosquamous cell carcinoma in the uterine cervix are described. Its cytologic and histologic findings are distinctive. The tumor cells had moderately amount and ground-glass cytoplasms, and had large nuclei containing a prominent nucleoli.