Essential DNA sequence for the replication of Rts1.

The promoter sequence of the mini-Rts1 repA gene encoding the 33,000-dalton RepA protein that is essential for replication was defined by RNA polymerase protection experiments and by analyzing RepA protein synthesized in maxicells harboring mini-Rts1 derivatives deleted upstream of or within the presumptive promoter region. The -10 region of the promoter which shows homology to the incII repeat sequences overlaps two inverted repeats. One of the repeats forms a pair with a sequence in the -35 region, and the other forms a pair with the translation initiation region. The replication origin region, ori(Rts1), which was determined by supplying RepA protein in trans, was localized within 188 base pairs in a region containing three incII repeats and four GATC sequences. Dyad dnaA boxes that exist upstream from the GATC sequences appeared to be dispensable for the origin function, but deletion of both dnaA boxes from ori(Rts1) resulted in reduced replication frequency, suggesting that host-encoded DnaA protein is involved in the replication of Rts1 as a stimulatory element. Combination of the minimal repA and ori(Rts1) segments, even in the reverse orientation compared with the natural sequence, resulted in reconstitution of an autonomously replicating molecule.     

Roles of the TGACT repeat sequence in the yeast TRP5 promoter

Yeast genes under general amino acid control contain multiple copies of a sequence known as the TGACT repeat in the 5'-flanking DNA. The yeast TRP5 gene contains two copies of the TGACT repeat sequence in its 5'-flanking region. The upstream TGACT repeat of TRP5 is required for normal basal expression as well as derepression by general control. Synthetic oligonucleotides containing a TGACT sequence were inserted into previously constructed TRP5 control region deletion mutants. A synthetic 17-base pairs (bp) oligonucleotide containing a TGACT copy along with flanking nucleotides from HIS3 was able to restore derepression in all deletion mutants tested. The 17-bp oligonucleotide also functioned bidirectionally. Replacements in the upstream control region by synthetic oligonucleotides indicated that sequences other than the TGACT repeat are required for high basal expression. Replacements of the downstream repeat sequence by the 17-bp oligonucleotide suggest its main role in this position is for derepressed expression. High level derepressed expression was found to correlate with the presence of two repeats.     

A centromeric tandem repeat family originating from a part of Ty3/gypsy-retroelement in wheat and its relatives

From a wild diploid species that is a relative of wheat, Aegilops speltoides, a 301-bp repeat containing 16 copies of a CAA microsatellite was isolated. Southern blot and fluorescence in situ hybridization revealed that approximately 250 bp of the sequence is tandemly arrayed at the centromere regions of A- and B-genome chromosomes of common wheat and rye chromosomes. Although the DNA sequence of this 250-bp repeat showed no notable homology in the databases, the flanking or intervening sequences between the repeats showed high homologies (>82%) to two separate sequences of the gag gene and its upstream region in cereba, a Ty3/gypsy-like retroelement of Hordeum vulgare. Since the amino acid sequence deduced from the 250 bp with seven CAAs showed some similarity ( approximately 53%) to that of the gag gene, we concluded that the 250-bp repeats had also originated from the cereba-like retroelements in diploid wheat such as Ae. speltoides and had formed tandem arrays, whereas the 300-bp repeats were dispersed as a part of cereba-like retroelements. This suggests that some tandem repeats localized at the centromeric regions of cereals and other plant species originated from parts of retrotransposons.     

Cloning and nucleotide sequence of frpC, a second gene from Neisseria meningitidis encoding a protein similar to RTX cytotoxins

Neisseria meningitidis FAM20 has recently been shown to produce two Fe-regulated proteins (FrpA and FrpC) related to the RTX family of cytotoxins. Here we report the cloning and DNA sequence of the locus containing the gene encoding the larger meningococcal RTX protein FrpC. FrpC was highly similar to FrpA throughout much of the predicted protein, with two main differences. Whereas the FrpA protein had 13 copies of the nine-amino-acid repeat units typical of RTX proteins, FrpC had 43 copies. The additional copies in FrpC apparently arose from a threefold tandem amplification of a 600bp DNA fragment encoding the repeats. In addition, the frpC gene lacked good promoter consensus sequences. An open reading frame (0RF1) of unknown function was found immediately upstream of frpC, suggesting the possibility that frpC was cotranscribed with ORF1. A probable promoter was found 300 bp upstream of ORF1, and it contained a Fur protein-binding sequence found in the promoters of Fe-regulated Escherichia coli genes. DNA upstream of the ORF 1/frpC promoter was homologous to IStO76-like elements surrounding capsulation loci of strains of Haemophilus influenzae. A FrpC-like protein (reactive in immunoblots with monoclonal antibody 9D4; multiple reactive bands of about 200 to 120kDa) was found in five out of eight meningococcal strains but only in one out of 14 other Neisseria, suggesting that FrpC may participate in the pathogenesis of meningococcal disease.     

Two integrated partial repeats of simian virus 40 together code for a super-T antigen.

We determined that the coding sequence for a 100-kilodalton super-T antigen found in Simian virus 40 mouse transformants spanned two separate partial repeats of the viral genome. The downstream repeat contained a complete Simian virus 40 large-T-antigen gene, whereas the upstream repeat was a truncated copy of the same gene. When the repeats were separated by subcloning, the capacity to code for the super-T antigen was lost. A small insertion or deletion in the origin-control region which preceded the second repeat could also destroy the ability to code for the 100-kilodalton protein. Our data suggest that differential splicing between parts of two gene copies was responsible for the additional molecular weight of this super-T antigen.     

A variant tandemly repeated nucleotide sequence in Balbiani ring 2 of Chironomus tentans

pCtBR2-2 is a genomic clone from Chironomus tentans that hybridized in situ to Balbiani ring 2 (BR2) on salivary gland polytene chromosome IV. DNA sequencing indicated that the insert contained nearly four copies of a 180 bp tandemly repeated nucleotide sequence that was distinctly different from a previously reported BR2 repeat. Sequence titration experiments detected about 70 copies of the 180 bp repeat per haploid genome, which would correspond to approximately 34% of a 37 kb BR2 gene. Each 180 bp repeat included a conserved 90 bp segment whose sequence was internally nonrepeating (INR), and a variable 90 bp repeated (R) segment comprised of three 30 bp repeats that may have evolved from a 9 bp consensus sequence. Results presented here raise the distinct possibility that other BR genes may contain significantly different repeated sequences that have not been identified.