DNA Coding For rRNA Research Articles

PRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants

To improve the selection phenotype of the expression plasmid pTEX, a Trypanosoma cruzi rDNA (DNA coding for rRNA) gene spacer fragment (806 bp) containing a mapped transcription start point ( tsp) was cloned in the vectors pTEX and pTEX- cat, generating the plasmids pRIBOTEX and pRIBOTEX- cat. T. cruzi cultures transiently transfected with pRIBOTEX- cat expressed a chloramphenicol (Cm) acetyltransferase (CAT) activity 16 000-fold greater than the activity observed with the parental vector pTEX- cat. Moreover, T. cruzi cells transformed with pRIBOTEX and pRIBOTEX- cat exhibited logarithmic growth in the presence of Geneticin (G418) 2 weeks earlier than that observed with controls transformed with pTEX. The plasmid copy number in stably transformed trypanosomes was about 50-times higher in cultures transformed with pTEX- cat than in cells transformed with pRIBOTEX or pRIBOTEX- cat. However, the neo RNA steady-state level and the CAT activity observed among the stably transfected cultures showed only modest differences. Finally, it was found that the pRIBOTEX vector was not episomally maintained as pTEX, but integrated into a chromosome indistinguishable from the one encoding rRNA. These features make pRIBOTEX a useful tool for transfection and rapid expression of genes in T. cruzi.

A transformation system for the yeast Candida utilis: use of a modified endogenous ribosomal protein gene as a drug-resistant marker and ribosomal DNA as an integration target for vector DNA.

We have developed a transformation system for the yeast Candida utilis. A novel strategy was applied to construct the transformation system, since auxotrophic mutants which could be used as hosts for transformation are not available. A gene encoding the ribosomal protein L41 was cloned from C. utilis, which is sensitive to cycloheximide, and used as a marker gene conferring cycloheximide resistance after modification of its amino acid sequence. The marker gene was constructed by substitution of the proline codon at position 56 with the glutamine codon by in vitro mutagenesis, as it had been reported previously that the 56th amino acid residue of L41 is responsible for the cycloheximide sensitivity of various organisms (S. Kawai, S. Murao, M. Mochizuki, I. Shibuya, K. Yano, and M. Takagi, J. Bacteriol. 174:254-262 1992). The ribosomal DNA (i.e., DNA coding for rRNA) of C. utilis was also cloned and used as a multiple-copy target for the integration of vector DNA into the genome, which resulted in a high transformation efficiency. Transformants were obtained by electroporation with a maximum efficiency of approximately 1,400 transformants per 1 microgram of linearized DNA carrying the gene for cycloheximide resistance and part of the ribosomal DNA. No transformants were obtained with intact plasmids. Multiple copies of the linearized plasmid were integrated into the host chromosome by homologous recombination. Southern analysis of the transformants in which vector DNA was integrated at the L41 gene locus indicated that there are two copies of gene for the L41 protein per cell, suggesting that C. utilis is diploid. Transformants were obtained from a variety of C. utilis strains, indicating that this method is applicable to the transformation of other C. utilis strains, even though there is significant heterogeneity in chromosomal karyotypes among these strains.

A rapid method for extraction and purification of DNA from dental plaque.

A rapid method based on previously described DNA extraction procedures was developed for the isolation of DNA from dental plaque samples. The isolated DNA is suitable for use in the PCR. Freeze-thawing, cell wall-degrading enzymes, and guanidine isothiocyanate were used to lyse cells and release DNA. The released DNA was adsorbed onto diatomaceous earth and purified by washing with guanidine isothiocyanate, ethanol, and acetone. The purified DNA was released from the diatomaceous earth into an aqueous buffer and analyzed by PCR with 16S rDNA primers (rDNA is DNA coding for rRNA). As judged from studies with pure cultures of a number of bacterial species, gram-negative and gram-positive organisms were lysed equally well by this procedure. The amount of PCR product was proportional to the number of cells analyzed over the range tested, 500 to 50,000 cells. On the basis of studies with plaque samples that were spiked with known quantities of the oral bacterium Treponema denticola, the DNA prepared from plaque was free of substances inhibitory to PCR. This method should have utility in molecular genetic studies of bacterial populations not only in uncultured plaque samples but also in other complex bacterial assemblages.

Prevalence of Borrelia burgdorferi sensu lato-infected ticks on migrating birds.

The prevalence of Lyme disease Borrelia-infected ticks on migrating birds was studied in Scandinavia. A total of 22,998 birds were caught at eight different bird observatories and examined for ticks. Five different species of ticks were found infesting the birds. The dominant species, Ixodesricinus, constituted 98.3% of the ticks collected. The presence of spirochetes was determined by an immunofluorescence assay of tick larvae and DNA amplification by PCR on all ticks. To determine which Borrelia burgdorferi sensu lato species were present, a species classification was performed by DNA amplification with species-specific 16S rDNA primers and by DNA sequencing (rDNA is DNA coding for rRNA). Flagellin gene sequences of all species of B. burgdorferi sensu lato previously recorded in Europe were observed. Borrelia garinii was the most prevalent Lyme disease Borrelia species in ticks collected from birds arriving from the South or Southeast in the spring, whereas the distribution was more heterogeneous in ticks from birds migrating from the Southwest. These data support the notion that birds are partly responsible for the heterogeneous distribution of Lyme disease Borrelia spirochetes in Europe.

The Trypanosoma cruzi ribosomal RNA-encoding gene: analysis of promoter and upstream intergenic spacer sequences

The transcription start point ( tsp) of the ribosomal RNA(rRNA)-encoding gene of Trypanosoma cruzi was mapped at 1550 bp upstream from the 18S rRNA coding sequence. The +1 nucleotide ( tsp) was determined to be a guanosine. As described for other eukaryotes, no consensus sequence was found when the putative promoter sequence (— 200 to + 50) was compared with that described for Trypanosoma brucei and Crithidia fasciculata. However, a repeated element was found in the upstream intergenic spacer sequence ( IGS) of T. cruzi. Motifs, present in this element, exhibit significant homology to the T. cruzi promoter sequence. Furthermore, the same motifs could be found, in a similar sequence organization, within the T. brucei promoter region. Therefore, the data described in this paper strongly indicate that the IGS rDNA (DNA coding for rRNA) organization in trypanosomatids appears similar to that found in higher eukaryotes.

Differences in the hybridization pattern of Bacillus subtilis genes coding for rDNA depend on the method of DNA preparation.

Three different methods of DNA isolation (organic deproteinization, potassium acetate deproteinization, and the use of cetyltrimethylammonium bromide) have been used to prepare DNA from Bacillus subtilis. Subsequent hybridization with an rDNA probe (DNA coding for rRNA) produces different patterns, which mirror those previously reported to indicate an rDNA deletion.

Length heterogeneity of amplified circular rDNA molecules in oocytes of the house cricket Acheta domesticus (Orthoptera: Gryllidae)

Amplification of the genes coding for rRNA occurs in the oocytes of a wide variety of organisms. The amplification process appears to be mediated through a rolling-circle mechanism. The approximate molecular weight of the smallest rDNA circles is equivalent to the estimated combined molecular weight of DNA which codes for a single ribosomal RNA precursor molecule and an associated non-transcribed spacer DNA sequence. RNA-DNA hybridization studies carried out on oocytes of the house cricket, Acheta domesticus, suggest that DNA coding for rRNA accounts for only a small fraction of the rDNA satellite, all of which is amplified in the oocyte. In order to test the possibility that the remainder of the amplified rDNA represents spacer and to determine whether a rolling-circle mechanism might also be involved in amplification in A. domesticus oocytes, rDNA was isolated from ovaries of A. domesticus and spread for electron microscopy. A large proportion of the rDNA isolated from ovaries is circular, while main-band DNA and rDNA prepared from other tissues demonstrates few if any circles. The mean size of the smallest rDNA circles is approximately 8 times longer than the length estimated for DNA which codes for 18S and 28 S rRNA. Denaturation mapping shows the rDNA circles to contain two major readily denaturing regions located about equidistant from one another on the circle. Each readily denaturing region accounts for 4--6% of the total DNA in the circle. The fact that only 12% of the average molecule is required to code for A. domesticus 18S and 28S rRNA is consistent with the hybridization data. Considerable size heterogeneity exists in the length of the smallest class of rDNA molecules. In the rDNA of other species such heterogeneity has been shown to reside in the non-transcribed spacer.

Absence of amplification of ribosomal DNA in the polytrophic meroistic ovary of the giant silkworm moth,Antheraea pernyi (Lepidoptera: Saturniidae).

In the meroistic insect ovary, the oocyte synthesizes little if any RNA. Most of the RNA which accumulates in the oocyte is synthesized by trophocytes. In the polytrophic meroistic ovary each oocyte is associated with a cyst containing 1,3,7 or 15 trophocytes. The trophocytes are derived from the same cell as the oocyte. The trophocyte cysts and the oocytes of the giant silkworm moth,Antheraea pernyi (Lepidoptera: Saturniidae), are large enough to enable their isolation by microdissection. The nucleus of each trophocyte is highly polyploid, containing hundreds of nucleoli. In order to determine whether DNA coding for rRNA (rDNA) is amplified in trophocytes ofA. pernyi, the percentage of the genome hybridizing with rRNA in somatic tissues was compared to that percentage in gametogenic tissues. RNA-DNA hybridization analysis indicates that approximately the same proportion (0.018%) of the DNA extracted from male and female gemetogenic tissues (testis, isolated trophocytes, and isolated oocytes) and somatic tissues (brain, Malpighian tubules) hybridizes with rRNA. The fact that DNA hybridizing with rRNA comprises the same proportion of the total DNA extracted from trophocytes, spermatogenic cells, and male and female somatic cells indicates that rDNA is not amplified in the trophocytes ofA. pernyi. In the polytrophic ovary, polyploidization of the entire trophocyte genome rather than amplification of a small part of it accounts for the increase of rDNA available for transcription.