Differential replication and DNA elimination in the polytene chromosomes of Euplotes crassus.

The transposon-like Tec elements of Euplotes crassus are precisely excised during formation of polytene chromosomes in the developing macronucleus. To determine whether all Tec elements exhibit identical developmental timing of excision, we used polymerase chain reaction to visualize amplification and diminution at numerous randomly selected Tec insertion sites. Two classes of sites are evident. Early replicating sites show one or more rounds of amplification and diminution (corresponding to excision) and frequently occur within macronuclear-destined sequences. Late replicating sites do not undergo diminution until chromosome fragmentation and are predominantly associated with eliminated sequences. We conclude that the previously described clustering of macro-nuclear-destined sequences in the micronuclear genome allows for their differential replication at the polytene stage and results in targeting of these sequences for transcriptional activation and highly specific deletion and chromosome fragmentation processes.

Structures of the Euplotes crassus Tec1 and Tec2 elements: identification of putative transposase coding regions.

The Tec1 and Tec2 transposon-like element families of Euplotes crassus are highly unusual in that all 30,000 copies of each family are excised from the genome during a discrete time period of macronuclear development. Complete nucleotide sequences were generated for the Tec1-1 and Tec2-1 elements, representing the Tec1 and Tec2 families. Open reading frames (ORFs) are conserved in position and sequence between the two elements, although sequences that comprise one ORF (ORF2) of Tec1-1 are split into two overlapping ORFs (ORFs 2A and 2B) in Tec2-1. ORF1 in Tec1-1, its homolog in Tec2-1 and one of the overlapping ORFs from Tec2-1 (ORF2B) contain TGA codons, which may be translated as Cys, as observed for two other Euplotid genes. Sequence analyses of ORFs from other members of each element family indicate that the families are distinct from each other and are highly conserved within each family. Computer searches of sequence databases have revealed sequence similarity between Tec ORF1s and the previously described Tc1-IS630 family of transposases which includes ORFs from bacterial, nematode and insect transposons.

para-aminobenzoate synthesis from chorismate occurs in two steps.

Escherichia coli p-aminobenzoate synthase is composed of two nonidentical subunits encoded by pabA and pabB and has been assumed to be the sole enzyme responsible for p-aminobenzoate biosynthesis from chorismate and glutamine. Plasmids were constructed that overproduce the p-aminobenzoate synthase subunits 250-500-fold. Partial purification of the subunits revealed that they form a diffusible intermediate that is subsequently converted to p-aminobenzoate by a second enzyme (Mr = 49,000) temporarily designated enzyme X.

Chromosomal organization and expression of Escherichia coli pabA.

The pabA gene in Escherichia coli and Salmonella typhimurium encodes the glutamine amidotransferase subunit of para-aminobenzoate synthase, which catalyzes the first reaction in the conversion of chorismate to para-aminobenzoate (PABA). We have determined the nucleotide sequences of 1,362 base pairs preceding E. coli pabA and of 981 base pairs preceding S. typhimurium pabA. The nucleotide sequences suggest the presence of two protein-coding regions immediately upstream of pabA, designated orf1 and fic. Transcription analysis indicates that E. coli pabA is encoded by two overlapping transcriptional units. The polycistronic transcriptional unit includes orf1-fic-pabA and is initiated by the promoter designated P2. The monocistronic unit includes only pabA and is initiated by the promoter designated P1, which is located in the fic-coding region. Both promoters transcribe pabA to about the same steady-state level. However, expression analysis using chromosomal pabA-lacZ translational fusions indicated that P1 expressed PabA at least 50-fold more efficiently than P2. pabA-dependent growth rate analysis indicates that P1 is essential and P2 is dispensable for PABA metabolism. In the absence of P1, growth was reduced as a result of insufficient PabA expressed from P2. The significance of these results and possible posttranscriptional control mechanisms which affect PabA expression from the P2-initiated polycistronic unit are discussed.

Characterization of mutations contributing to sulfathiazole resistance in Escherichia coli.

A sulfathiazole-resistant dihydropteroate synthase (DHPS) present in two different laboratory strains of Escherichia coli repeatedly selected for sulfathiazole resistance was mapped to folP by P1 transduction. The folP mutation in each of the strains was shown to be identical by nucleotide sequence analysis. A single C-->T transition resulted in a Pro-->Ser substitution at amino acid position 64. Replacement of the mutant folP alleles with wild-type folP significantly reduced the level of resistance to sulfathiazole but did not abolish it, indicating the presence of an additional mutation(s) that contributes to sulfathiazole resistance in the two strains. Transfer of the mutant folP allele to a wild-type background resulted in a strain with only a low level of resistance to sulfathiazole, suggesting that the presence of the resistant DHPS was not in itself sufficient to account for the overall sulfathiazole resistance in these strains of E. coli. Additional characterization of an amplified secondary resistance determinant, sur, present in one of the strains, identified it as the previously identified bicyclomycin resistance determinant bcr, a member of a family of membrane-bound multidrug resistance antiporters. An additional mutation contributing to sulfathiazole resistance, sux, has also been identified and has been shown to affect the histidine response to adenine sensitivity displayed by these purU strains.