In vivo expression of single-stranded DNA in mammalian cells with DNA enzyme sequences targeted to C-raf.

The use of antisense oligodeoxynucleotides (AS-ODN) remains a viable method to downregulate selected gene function. However, limitations to the antisense approach remain, such as (1) difficulties in delivery of the AS-ODN into target tissues, (2) instability of AS-ODN in vivo, (3) uncertanties about the precise mode of action, and (4) toxic effects in animal and human studies. To circumvent some of these difficulties, we designed a vector set that directs the in vivo production of single-stranded DNA (ssDNA) of a desired target sequence with limited extraneous vector nucleotide sequences. One plasmid was designed to express Moloney murine leukemia virus (MoMuLV) reverse transcriptase (RT). Another expression plasmid contains the MoMuLV primer binding site at the 3'-end of its RNA transcript so that an ssDNA would be synthesized by RT when both plasmids are cotransfected into cells. To test this expression system, we constructed a plasmid set, pssXA/pssXB that produces ssRNA-cleaving DNA 10-23 enzyme (Santoro, S.W., and Joyce, G.F. [1997]. Proc. Natl. Acad. Sci. USA 37, 13330-13342). The DNA enzyme sequence was placed between two oligonucleotide arms that are complementary and able to specifically target C-raf kinase mRNA. These plasmids were transfected into the A549 lung carcinoma cell line. Reduced C-raf mRNA levels by up to 34%-36%, as determined by Northern blot analysis, were observed in the transfected cells. Our results demonstrate the feasibility of using this novel ssDNA expression system to generate any sequence of interest in vivo for antisense, RNA-cleavage DNA enzyme, or triplex-forming strategies.

Identification of a functional CT-element in the Phytophthora infestans piypt1 gene promoter.

CT-rich sequences of incompletely characterized function have been found in the gene promoter regions of many organisms, fungi and members of the genus Phytophthora prominently among them. We describe here an in vitro analysis of CT-element function in regulating transcription of the Phytophthora infestans piypt1 gene, a gene that encodes a monomeric G-protein believed to be involved in regulation of vesicle transport (Chen and Roxby (1996) Gene 181, 89-94). The results of the promoter analysis indicate that a 17-bp CT-element lying close to the transcription start point of this gene is important in determining the frequency of transcription initiation. Competition experiments suggest that transcription factors bind to the CT element. A subregion lying at the 5'-end of the CT-element resembles an Inr element, a type of CT-rich transcription regulator first discovered in some mammalian genes. This Inr-like subregion appears to be more important in the interactions leading to transcription initiation than more downstream regions within this CT-element. Two proteins, of 37 and 45 kDa, respectively, that bind to the CT-element and are presumed to be transcription factors were detected in P. infestans nuclear extracts by southwestern blotting.

Population structure of Mya arenaria along the New England coastline.

Sequences of the internal transcribed spacer (ITS-1) ribosomal DNA region were compared among 88 soft-shell clams (Mya arenaria) from 12 sites (within three general areas) along the New England coast to determine whether populations were genetically heterogeneous. Two sequence variants were observed, with type 1 having a 3-nucleotide insertion and one point mutation relative to type 2. Allele-specific polymerase chain reaction (PCR); using primers specific to each sequence type, was performed to determine the distribution of individuals who had both allelic forms. DNA from soft-shell clams collected from three areas (Cobscook Bay, Maine; Gulf of Maine; and southern New England) were compared chi 2 analyses of allele-specific PCR results revealed no significant heterogeneity among the three population distributions.

Characterization of a Phytophthora infestans gene involved in vesicle transport.

Members of the Ras superfamily of monomeric GTP-binding proteins have been shown to be essential in specific steps of vesicle transport and secretion in widely divergent organisms. We report here the characterization of a gene from Phytophthora infestans encoding a deduced amino acid (aa) sequence belonging to the Ypt class of monomeric GTP-binding proteins, products shown in other organisms to be essential for vesicle transport between the endoplasmic reticulum and the cis-Golgi compartments. Analysis of genomic and cDNA sequences of this gene, Piypt1, indicates that it contains five introns, one in the 5'-untranslated region. All introns are typical in beginning with GT and ending with AG. The region of the transcription start point displays a number of features characteristic of fungi and other eukaryotes, but it does not contain TATA or CAAT motifs. A single transcript is produced from the gene, which is polyadenylated, but the gene does not contain a recognizable polyadenylation signal. Genomic DNA blots indicate that Piypt1 is a single-copy gene. Comparisons of Ypt1 aa sequences indicate that P. infestans is more closely related to algae and higher plants than to the true fungi. The protein product of the Piypt1 gene, expressed in Escherichia coli, cross-reacts with antiserum against yeast Ypt1 protein and binds GTP. Furthermore, the Piypt1 gene is able to functionally complement a mutant ypt1 gene in Saccharomyces cerevisiae. The aa sequence similarity, immunological cross-reactivity and functional attributes of Piypt1 make it likely that it is an authentic ypt1 gene which participates in vesicle transport in Phytophthora infestans.

Physical properties of a plasmid-like DNA from Euglena gracilis.

A small circular extrachromosomal DNA of the flagellate protozoan Euglena gracilis has been characterized as having a contour length of 11.3 kb, with a consistent restriction map. The buoyant density (rho = 1.717) and melting temperature (tm = 89 degrees C) both indicate a base content of 59% G + C. The DNA is found in both wild-type cells and those lacking plastids. The copy number is estimated to be about 1000.

Effects of ultrasound on catalase and malate dehydrogenase.

Catalase and malate dehydrogenase (MDH) were subjected to the sound field produced by a transversely oscillating wire driven at 20 kHz. Catalase was not inactivated under any conditions of sonication whereas MDH inactivation increased exponentially with the duration of sonication and depended upon the initial enzyme concentration. The inactivation was not the result of collapse cavitation or thermal inactivation and was probably related to the presence of acoustic microstreaming.

Molecular weights of aggregation states of Busycon hemocyanin.

Molecular weights of all hemocyanin aggregates which can be homogeneously isolated have been measured by sedimentation equilibrium. The larger aggregates, which are the ones present under physiological conditions, are, to a very close approximation, integral multiples of a 4.4 x 10(6)-dalton, 60 S species. Dissociation of the 60 S species at high pH gives heterogeneous samples in which the smallest species has a molecular weight of 300,000. The smallest subunit which can be produced in denaturing solvents also has a molecular weight of 300,000.