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We report the isolation and characterization of two new genomic loci corresponding to the mouse Dishevelled (Dvl) genesDvl2 and Dvl3. The Dvl genes are homologs of the Drosophila dsh segment polarity gene, and are involved in theWnt/wingless signal transduction pathway. Dvl2 and Dvl3 genomic clones were isolated from a mouse 129 strain λFIXII genomic library and have identical exon/intron organization...
The subunit composition of RNA polymerase II (polII) was compared between the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. For this purpose, we partially purified the enzyme from S. pombe. Judging from the co-elution profiles in column chromatographies of both the RNA polymerase activity and the two large subunit polypeptides (subunit 1 (prokaryotic β ...
The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown...
Fibroblast growth factor 1 (FGF-1 or aFGF), is the prototype member of the heparin-binding growth factors which are capable of angiogenesis in vivo. FGF-1 has been implicated in atherosclerosis, cancer, wound repair and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the etiopathogenesis of inflammation and cancer, we have undertaken steps to isolate and...
Plasmids have been constructed that allow integration by a double recombination event at the thrC locus of the Bacillus subtilis (Bs) chromosome. These plasmids can be used either for construction of merodiploid strains and complementation analysis, or for construction of transcriptional fusions to the Escherichia coli lacZ gene. The plasmids contain an antibiotic (An) marker selectable in Bs, as...
The postnatal increase in skeletal α-actin (Sk-α-Act) synthesis in pigs is due, in part, to increased transcription. To characterize the factors responsible for its transcriptional regulation, we have cloned and determined the nucleotide sequence of a 5.2-kbHind III genomic DNA fragment which contains the complete coding region of Sk-α-Act, distributed over seven exons, plus 1.9 kb of 5 flanking...
The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited...
We have developed an Escherichia coli system for testing the behaviour of plasmids carrying target sites for the Flp site-specific recombinase. The E. coli strain BL-FLP is described, which carries a chromosomally integrated bacteriophage T7 RNA polymerase gene expressed from a lac promoter, and harbours the plasmid pMS40. pMS40 has the features: (i) it carries the FLP recombinase gene under the...
Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC. The role of the cyaC gene product in the acylation...
The genomic organization of the gene encoding rat aspartyl-tRNA synthetase (AspRS), a class II aminoacyl-tRNA synthetase (aaRS), was determined. A single active gene and several pseudogenes were isolated from a rat genomic DNA library and characterized. The active DRS1 gene encoding the rat AspRS spans approximately 60 kb and is divided into 16 exons. Exons 8-16, encoding the nt-binding domain...
The macrolide antibiotic, tylosin (Ty), is produced by Streptomyces fradiae. Two resistance determinants (tlrA, synonymermSF , and tlrD) conferring resistance to macrolide, lincosamide and streptogramin B type (MLS) antibiotics were previously isolated from this strain, and their products shown to methylate 23S ribosomal RNA (rRNA) at a common site, thereby rendering the ribosomes MLS resistant....
Among the small nuclear RNAs (snRNAs) involved in the spliceosomal processing of pre-mRNA, U6 is the most conserved. As a first evidence for the presence of the splicing machinery in the amitochondrial protozoan Entamoeba histolytica (Eh), we have cloned the u6 snRNA gene. We find that in this organism u6 is a single copy gene that is transcribed as a poly(A) - RNA molecule of approximately...
The cyclin-dependent kinase inhibitor p27 Kip1 plays an important role in regulating cell-cycle progression. p27 Kip1 directly inhibits the catalytic activity of cyclin/cdks (cyclin-dependent kinase) complexes and/or interferes physically with cyclin/cdks activation by CAK. Interestingly, the expression level of p27 Kip1 mRNA was...
A DNA sequencing strategy was developed based on the tetracycline resistance transposon Tn1721. A universal M13 primer binding site (UP) for DNA sequencing and restriction sites for mapping were inserted near one end of Tn1721 and the new derivative, Tn5491, introduced onto a conjugative F plasmid. The target sequence is inserted between two inverted resolution sites (res) of Tn1721 present on...
We report the isolation of a large cyclophilin protein containing RS (arginine-serine) repeats from a yeast two-hybrid screen using Clk (CDC28/cdc2-like kinase) as a probe. This Clk associating RS-cyclophilin (CARS-Cyp) possesses 39% homology to the NK-TR 1 (natural killer tumor recognition protein-1) we have previously characterized (Anderson et al. (1993) Proc. Natl. Acad. Sci. USA 90 (1993)...
A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA andfemB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed inSa . Intra- and interspecies relatedness of these...
The galactose operon encoding a repressor and genes for the Leloir pathway for galactose metabolism (galactokinase, galactose-1-phosphate-uridyl transferase and UDP glucose-4-epimerase) was located adjacent to the multiple sugar metabolism (msm) operon on the chromosome of Streptococcus mutans Ingbritt (serotype c) and the complete nucleotide sequence of this 5-kilobase region was determined. The...
SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5 -GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase...
The expression of the hepatic lipase (HL) gene is highly tissue specific. In order to identify cis-acting elements which regulate the expression of this gene in the liver, multiple deletion mutants of the 5 -flanking region of the HL gene fused to the human growth hormone gene were transfected in HepG2 cells, which normally produce HL. Transient expression assays indicated the presence of negative...
A hyperthermophile NC12 was newly isolated from Noboribetsu hot spring. To characterize this organism, a gene coding for 16S rRNA was cloned and sequenced. The 16S rRNA sequence from NC12 shows the highest similarity with those from Pyrodictium occultum and Desulfurococcus mobilis among the sequences in the database, indicating that NC12 belongs to a cluster of extreme thermophiles (Crenarchaeota)...
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