Cloning and chromosomal mapping of the mouse DNA-dependent protein kinase gene

 Severe combined immune deficiency (scid) mice are assumed to have two types of abnormalities: one is high radiosensitivity and the other is abnormal recombination in immunoglobulin and T-cell receptor genes. The human chromosome 8 q1.1 region has an ability to complement the scid aberrations. Moreover, the localization of the subunit DNA-dependent protein kinase [DNA-PK_cs] participating in DNA double-strand break repair in the same locus was clarified. In scid mouse cells, the number of DNA-PK_cs products and extent of DNA-PK activity remarkably decrease. These observations gave rise to the assumption that DNA-PK_cs is the scid factor itself. In order to determine whether the DNA-PK _cs gene is the scid gene, we isolated the mouse DNA-PK _cs gene and investigated its chromosomal locus by fluorescence in situ hybridization (FISH). Consequently, it became clear that the mouse DNA-PK _cs gene existed in the centromeric region of mouse chromosome 16, determined by cross-genetic study, as a scid locus. This finding strongly suggests that mouse DNA-PK _cs is the scid gene. Received: 22 March 1996     

Structure, function and expression of a murine homeobox protein AREC3, a homologue of Drosophila sine oculis gene product, and implication in development.

The cDNA clones encoding ARE(Na,K-ATPase alpha1 subunit gene regulatory element) binding protein AREC3 were isolated from myoblast C2C12 cells and mouse skeletal muscle cDNA library. At least four alternatively spliced forms of AREC3 cDNA were identified. Sequence analysis indicates that AREC3 has an extensive homology with the Drosophila sine oculis gene product required for development of the entire visual system [Cheyette et al.(1994) Neuron 12, 977-996]. The homologous region including a homeodomain is required for specific DNA binding to ARE. A transactivation domain was identified in the C-terminal part of the AREC3 by reporter gene assays using GAL4-AREC3 fusion protein constructs. Immunohistochemistry revealed that AREC3 localized to the nucleus and cytoplasm of myoblast C2C12 cells, and the production of AREC3 is augmented during muscle differentiation. Western blot analysis indicated that the 115 kDa form of AREC3 protein is increased in the cytoplasmic extract, and the 67kDa form is increased both in nuclear and cytoplasmic extracts of C2C12 cells during muscle differentiation.     

Regulation of choline acetyltransferase/iacZ fusion gene expression in putative cholinergic neurons of drosophila melanogaster

We have analyzed the distribution of putative cholinergic neurons in whole-mount preparations of adult Drosophila melanogaster. Putative cholinergic neurons were visualized by X-gal staining of P-element transformed flies carrying a fusion gene consisting of 5′ flanking DNA from the choline acetyltransferase (ChAT) gene and a lacZ reporter gene. We have previously demonstrated that cryostat sections of transgenic flies carrying 7.4 kb of ChAT 5′ flanking DNA show reporter gene expression in a pattern essentially similar to the known distribution of ChAT protein. Whole-mount staining of these same flies by X-gal should thus represent the overall distribution of ChAT-positive neurons. Extensive staining was observed in the cephalic, thoracic, and stomodeal ganglia, primary sensory neurons in antenna, maxillary palps, labial palps, leg, wing, and male genitalia. Primary sensory neurons associated with photoreceptors and tactile receptors were not stained. We also examined the effects of partial deletions of the 7.4 kb fragment on reporter gene expression. Deletion of the 7.4 kb fragment to 1.2 kb resulted in a dramatic reduction of X-gal staining in the peripheral nervous system (PNS). This indicates that important regulatory elements for ChAT expression in the PNS exist in the distal region of the 7.4 kb fragment. The distal parts of the 7.4 kb fragment, when fused to a basal heterologous promoter, can independently confer gene expression in subsets of putative cholinergic neurons. With these constructs, however, strong ectopic expression was also observed in several non-neuronal tissues. © 1995 John Wiley & Sons, Inc.     

K-ras Gene Mutation Related to Histological Atypias in Human Colorectal Adenomas

To investigate the relationship of oncogene analysis to morphology, we analyzed K-ras gene mutations by dot-blot hybridization with and without consideration of histological atypias in individual colorectal adenomas. Each of 54 colon polyps were divided into two parts after fixation. One part was used as a mass to assess point mutations; the remaining portion of each polyp was paraffin-embedded, stained with hematoxylin and eosin, and examined for point mutations related to histological atypias. In the first part of our study, K-ras gene mutations at codon 12 were detected in 13 cases (24%). In the second part of our study, 12 cases had distinctly different histological atypias. From each of these 12 cases, two areas, one with higher or one with lower grade atypia in the same polyp were excised to analyze for K-ras gene mutation. Two of these 12 cases (17%) had the mutation in different areas of the same tumor. These two cases contained the mutation only in the areas with higher grade atypia, and only one case added information regarding ras mutation upon microdissection when compared to the entire biopsy. These results suggest that oligonucleotide hybridization can identify the majority of cases containing ras mutations despite regional morphologic variation. Individual cases, however, may contain clonal subpopulations within adenomas with different ras sequences from other regions within the same adenoma.     

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.     

Mutant isolation of mouse DNA topoisomerase II alpha in yeast.

For characterizing in vivo functions of a mammalian protein, it is informative to obtain conditional mutations and apply them to the mouse genetic system. However, the isolation of conditional mutations has been quite difficult in cultured cells. We report here that functional expression of a heterologous mammalian gene in the yeast Saccharomyces cerevisiae provides a system for isolating mutated genes. We found that the cloned mouse TOP2 alpha cDNA, which encodes mouse DNA topoisomerase II (topo II) alpha, could rescue the lethal phenotype caused by yeast top2 null mutation. In order to generate and select temperature-sensitive mouse topo II alpha, an expression plasmid was mutagenized in vitro and was transformed, using the plasmid shuffling method, into the yeast strain, in which the endogenous TOP2 gene had been disrupted. We observed that one of such clone of yeast cells harboring a mutagenized mouse TOP2 alpha showed temperature-sensitive growth. Enzymatic assays and sequencing analysis revealed that this phenotype was caused by the thermosensitive nature of the mutant mouse protein, which has isoleucine at amino acid 961 instead of threonine. Therefore we have isolated the first conditional mutation in the mouse TOP2 alpha.     

Characterization of spinach leaf α-l-arabinofuranosidases and β-galactosidases and their synergistic action on an endogenous arabinogalactan-protein

Two β-galaclosidases (β-Galase-I and -II, EC 3.2.1.23) and two α-l -arabinofuranosidases (α-l -Arafase-I and -II. EC 3.2.1.55). were purified from mesophyll tissues of spinach (Spinacia oleracea L.), using chromatography on DEAE-cellulose, lactose-conjugated Sepharose CL-4B, and Sephadex G-100, or on hydroxylapatite and Sephadex G-150. The apparent molecular mass (M_r) of β-Galase-I and -II, respectively, were estimated to be 38 000 and 58 000 on SDS-PAGE and 64 000 and 60 000 on gel-permeation chromatography, indicating that the former was a dimeric protein. The isoelectric points of β-Galase-I and -II were 6.9 and 5.2, respectively. Both enzymes hydrolyzed maximally p-nitrophenyl (PNP) β-galactoside at pH 4.3, and were activated about 2-fold in the presence of BSA (100 μg ml^?1). The activity of both enzymes was inhibited strongly by heavy metal ions and p-chloromercuriberszoate (p-CMB). d -Galactono-(1→4)-lactone and d -galactal served as potent competitive inhibitors for the enzymes. β-Galase-I and -II could be distinguished from each other in their relative rates and kinetic properties in the hydrolysis of aryl β-galactosides as well as of lactose and galacto-oligosaccharides. In particular. β-Galase-I exhibited a preferential exowise cleavage of β-1,6-galactotriose and β-1.3-galactan. α-l -Arafase-l (M_r 118000) and -II (M, 68 000) were optimally active on PNP α-l -arabinofuranoside at pH 4.8 and gave K_m values of 1.2 and 2.2 mM. respectively. l -Arabino-(1 → 4)-lactone. Ag⁺, and SDS acted as inhibitors for the isozymes. α-l Arafase-I was characterized by its activity to hydrolyze PNP β-d -xylopyranoside besides PNP α-l -arabinofuranoside. inhibition by d -xylose and d -glucono-(1 → 5)-lactone. and less sensitivity to Hg²⁺. Cu²⁺, and p-CMB. Sugar beet arabinan was hydrolyzed rapidly by α-l Arafase-II at one-half the rate for PNP α-l arabinofuranoside, while the polysaccharide was less susceptible to α-l Arafase-I. A spinach leaf arabinogalactan-protein was practically resistant to the action of β-Galases, but its susceptibility to the enzymes increased remarkably after prior hydrolysis with α-l Arafase-Il.     

A Serine Protease in Soybean Seeds That Acts Specifically on the Native {alpha} Subunit of {beta}-Conglycinin

A proteolytic activity directed against the     

Detection of Antibody-Coated Bacteria in Expectorated Sputum for Diagnosis of Lower Respiratory Infections

We evaluated antibody-coated bacteria (ACB) in expectorated sputum to discriminate contaminating or colonizing organisms from true pathogens. We examined 60 expectorated sputum samples from 51 patients with lower respiratory infections (chronic obstructive pulmonary disease 25, pneumonia 20, purulent tracheobronchitis 6). All samples were examined with quantitative culture and immunofluorescent demonstration of ACB. From the results of quantitative culture, we divided specimens into pathogen-isolated and pathogen-free samples. Among pathogen-isolated samples, in which we isolated accepted pathogenic organisms at ≥ 10⁷ colony-forming units per ml, 16 of 23 samples were ACB-positive (69.5%). In contrast, among pathogen-free samples, in which we isolated accepted pathogens at < 10⁷ colony forming units per ml or only upper respiratory flora, only 3 of 37 samples were ACB-positive (8.1%). The ACB-positive rate was significantly higher in pathogen-isolated than in pathogen-free samples (P < 0.001). Consequently, detecting ACB in expectorated sputum shows good potential as another criterion for distinguishing contaminating or colonizing organisms from true pathogens.     

Human T Cell Leukemia Virus Type 1 (HTLV-1) Antibodies in Healthy Populations and Renal Transplanted Patients in the North-East of Brazil

The seroprevalence of human T cell leukemia virus type 1 (HTLV-1) infection was investigated in Brazilians (570): native inhabitants (298) and descendants from Japanese (272) living in Recife and its neighborhoods—North-east of Brazil. Furthermore, polytransfused renal transplanted patients (54) were also examined for the serological status to this virus. The seropositivity to HTLV-1, screened by enzyme-linked immunosorbent assay (ELISA), was low: 1.34% for the local population and 0.73% for the descendants from Japanese. However, the seropositivity for the renal transplanted patients was found to be 11.1%. This higher value suggests that this retrovirus infection seems to be of importance in this clinical condition.