The nucleotide sequence of the entire histidine transport operon from Salmonella typhimurium has been determined and is shown to consist of four genes, hisJ, hisQ, hisM and hisP. This operon provides the only example of a binding protein-dependent transport system for which the total number of protein components is known. Determination of the amino acid compositions and sequences of these four transport proteins, together with analysis of various transport mutants, allows us to propose a molecular model for binding protein-dependent transport.

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11-to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two-to fourfold. These lines were resistant to PALA concentrations 20-to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five singlestep mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.Gene amplification is a major mechanism through which mammalian cells (2,12,21) and microorganisms (9) achieve resistance to a variety of anti-proliferative agents. Amplification also plays a role in normal development when there is need for very rapid synthesis of RNA, i.e., rRNAs in Xenopus laevis oocytes (5) and mRNAs for chorion proteins in Drosophila melanogaster follicle cells (19). Although the mechanisms differ greatly in each of these systems, detailed analyses of each may reveal common features.

Proteins on the merozoite surface of the human malarial parasite Plasmodium falciparum are targets of the host's immune response. The merozoite surface location of p75, a 75 kd P. fakciparum protein, was established by immunoelectron microscopy using antisera raised to the expressed product of a cDNA clone. Immunoprecipitation from protein extracts biosynthetically labeled during different periods of the asexual cycle showed that p75 is made continuously, although ringstage parasites appear to synthesize larger quantities. p75 is conserved and invariant in size in eight isolates of P. falciparum. The 880 bp cDNA sequence encoding part of p75 reveals one open reading frame containing a repetitive sequence unit of four amino acids. The predicted reading frame is correct since antisera to a synthetic peptide corresponding to the repetitive region recognize p75 in immunoblots. The sequence of p75 is homologous with the sequences of proteins from the ubiquitous, highly conserved family of 70 kd heatshock proteins, suggesting an important physiological function for p75. The cDNA fragment encoding part of p75 hybridizes with multiple genomic fragments, whose sizes are identical in DNA from nine P. falciparum strains, suggesting that the gene for p75 is well conserved and may be part of a gene family.

Mutations in the ack (acetate kinase) and pta (phosphotransacetylase) genes in Salmonella typhimurium were characterized and determined to be analogous to those of previously described Escherichia coli mutants. We established that in both bacterial species these genes were cotransducible with the neighboring histidine transport operon and were distally located relative to purF. pta mutants were sensitive to the dye alizarin yellow and were unable to grow on medium containing inositol as a carbon source. We selected mutants of both species with deletions covering both the ack and the pta genes; some deletions extended into the histidine transport operon. Two genetic loci, ack and pta, which encode the enzymes acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1 [reaction 1]) and phosphotransacetylase (acetyl-coenzyme A [CoA]: orthophosphate acetyltransferase, EC 2.3.1.8 [reaction 2]), respectively, which together synthesize acetylphosphate, were recently identified in Escherichia coli and mapped nearpurF, at 49 min on the linkage map (3-5). acetate + ATP ;± acetylphosphate + ADP (1) acetylphosphate + CoA.SH i± acetyl-CoA + Pi (2)