rRNA-encoding DNA Research Articles

Campylobacter species identification based on polymorphism of DNA encoding rRNA

Total DNA from five Campylobacter species was digested with a mixture of XhoI and BglII restriction endonucleases and analyzed by Southern hybridization by using a probe complementary to the DNA coding for the 16S rRNA. Each of the Campylobacter species, including C. jejuni, C. coli, C. laridis, C. fetus, and C. upsaliensis, displayed a characteristic pattern. Although some bands may be common to different species, the simplicity of the hybridization pattern enabled us to discriminate among the different species of Campylobacter.

Yeast artificial chromosomes with 200- to 800-kilobase inserts of human DNA containing HLA, V kappa, 5S, and Xq24-Xq28 sequences.

Sequences hybridizing to several human gene probes have been recovered as cloned inserts in yeast artificial chromosomes (YACs). Among 2300 YACs made from human leukocyte DNA (totaling about 0.1 genomic equivalent of human DNA) we have found two, 200 and 780 kilobases (kb), containing sequences of V kappa I immunoglobulin (V = variable); one, 240 kb, with class I HLA; and 11, 200-800 kb, with 5S rRNA-encoding DNA (rDNA). Fifty human YACs from a hamster-human cell hybrid with only the Xq24-Xq28 portion of the X chromosome include one that contains two anonymous probe sequences, DX13 and St14, previously inferred by indirect means to lie within about 70 kb of one another in Xq28. The YACs specific for human DNA arise at a frequency equivalent to the fraction of cellular DNA that is human-specific. Furthermore, the human YACs, formed in a 280-fold excess of hamster DNA, do not hybridize to a hamster DNA probe, indicating that individual YACs do not contain a combination of human and hamster DNA. To confirm that sequences are not scrambled, the YACs containing V kappa I or DX13 and St14 sequences were shown to produce restriction fragments identical in mobility to fragments detected by the same probes in total human DNA digested with the same enzymes. YACs may therefore provide large clones to bridge gene mapping at the chromosome level to molecular analyses of small fragments of genomic DNA.

In vivo double-strand breaks occur at recombinogenic G + C-rich sequences in the yeast mitochondrial genome.

An optional 46-base-pair G + C-rich element (GC cluster) in the coding region of the yeast mitochondrial var1 gene inserts preferentially in crosses into recipient alleles that lack the sequence. Unlike a similar gene conversion event involving the insertion of an optional 1143-base-pair intron, the mitochondrial 21S rRNA gene, which requires the action of a protein encoded by a gene within that intron, conversion of the var1 GC cluster does not require any protein product of the mitochondrial genome. We have detected double-strand breaks in the var1 gene in mitochondrial DNA isolated from unmated haploid rho+ and rho- strains at or near the boundaries of the optional GC cluster, as well as at a conserved copy of that sequence 160 base pairs upstream. No double-strand breaks were detected in the recipient var1 DNA molecules in the vicinity of the optional GC cluster target sequence. This contrasts with 21S rRNA-encoding DNA (rDNA) intron conversion where the recipient, but not the donor DNA, is cleaved at the element insertion site. These results suggest that although the 21S rDNA intron and the var1 GC cluster are preferentially inserted into their respective short alleles, these conversions probably occur by different mechanisms.

Glucocorticoid inhibition of initiation of transcription of the DNA encoding rRNA (rDNA) in lymphosarcoma P1798 cells.

Cell-free extracts of lymphosarcoma P1798 cell culture lines support faithful initiation upon the cloned mouse DNA encoding rRNA (rDNA) promoter, whereas extracts from cells treated for 16 hr with 0.1 microM dexamethasone cannot. Extracts from both sources transcribe the cloned 5S RNA gene in vitro and mixing experiments further demonstrate that inhibition of transcription of rDNA in vitro is not due to nucleases or inhibitors of transcription present in extracts from glucocorticoid-treated cells. Incubation of extracts from control cells at 45 degrees C for 15 min inactivates RNA polymerase I and abolishes transcription. Activity can be restored by the addition of partially purified RNA polymerase I from control cells and hormone-treated cells. Moreover, extracts from hormone treated cells can be reconstituted by the addition of a partially purified, heat-stable transcription factor from control cells.