The rat basophilic leukemia (RBL) cell lines were cloned and the various sublines compared for their chromosome number, IgE-mediated histamine release and for IgE surface receptors. It was found that cell lines started from tumors at different times vary in both their chromosome number and their ability to release histamine by an IgE-mediated reaction. RBL-I and III have approximately 44 chromosomes and did not respond to an IgE-mediated reaction. RBL-II and RBL-IV have 68-73 chromosomes and showed moderate levels of histamine release (percent release mean = 5 +/- 2 and 10 +/- 4, respectively). The cloning of the RBL-IV line resulted in some sublines which were excellent histamine releasers (range 39-100%) and some which were relatively refractory (less than 10%) to IgE-mediated histamine release. These clones did not differ significantly in chromosome number. Recloning the releasing lines gave rise to poor releasers, whereas the recloning of poor releasers did not produce good releasers indicating that the mutational drift in culture is toward loss of histamine-releasing capacity. The number of IgE receptors and the rate of IgE association and dissociation were similar for the different cell lines. The study failed to disclose significant molecular weight differences in the IgE receptor from the various clones and sublines indicating that the failure to release probably does not reside in the receptor. The various cloned sublines are phenotypically stable, and the isolation of excellent histamine-releasing sublines are useful for studies of the complex phenomenon of the histamine release.

By Southern blotting and hybridization analysis using 32P-labeled poly(dT-dG)-poly(dC-dA) as a probe, we have found, in eukaryotic genomes, a huge number of stretches of dTdG alternating sequence, a sequence that has been shown to adopt the Z-DNA conformation under some conditions. This sequence was found in all eukaryotic genomes examined from yeast to human, indicating extraordinary evolutionary conservation. The number of the sequence ranged from about 100 in yeast to tens of thousands in higher eukaryotes. Comparison of nucleotide sequences of dT-dG alternating regions and its flanking regions in several cloned genes showed that the repeated element [the Z(T-G) element] consists only ofdT-dG alternating sequence with variable length. The presence of another purine-pyrimidine alternating sequence was also surveyed in eukaryotic genomes by Southern blot hybridization using 32P-labeled poly(dG-dC)-poly(dGdC) as the probe. The stretches of dC-dG alternating sequence [the Z(C-G) element] were found to be moderately repetitive in human, mouse, and salmon genomes. However, a few and no copies of the Z(C-G) element were found in yeast and calf genomes, respectively. These results provide evidence for the abundance of potential Z-DNA-forming sequences in nature.Recent physicochemical studies of DNA conformation have shown that some synthetic DNAs with certain primary sequences have a novel conformation, called the Z form (1-4). Although the Z conformation was first observed with poly(dGdC) and most studies on Z-DNA have been done with it, other synthetic purine-pyrimidine alternating sequences such as poly(dT-dG)-poly(dC-dA) (2, 5, 6) and poly(ds4A-dT) (2) have also been shown to adopt the Z conformation. Until recently, however, there has been little direct evidence that such Z-DNA-forming sequences exist in native DNA. Nordheim et at (7) have shown that a specific antibody against brominated poly(dG-dC)-poly(dG-dC), a polymer that forms a Z-DNA under physiological conditions, reacts with interband regions of Drosophila polytene chromosomes. Recently, we have shown that the human genome has approximately 105 copies of stretches of dT-dG alternating sequence (8). A tandem block of 17 T-G (9) and 27 T-G dinucleotides (10) were found in human globin and in mouse immunoglobin genes, respectively, but the general occurrence of these sequences in the genomes was not investigated.Here we report that one of the Z-DNA-forming sequences, a long stretch of dT-dG alternating sequence is the sole unit of a repeated element [designated the Z(T-G) element] that is highly conserved throughout eukaryotic genome evolution. Furthermore, another Z-DNA-forming sequence, a stretch of dC-dG sequence, was found to be at least a part of another repeated element [designated the Z(C-G) element] and is moderately repeated in human, mouse, and salmon genomes but not in yeast or calf DNA.MATERIALS AND METHODS Materials. Calfthymus DNA, salmon sperm DNA, and poly-(dG-dC)-poly(dG-dC) were purchased from Sigma. Poly(dTdG)-poly(dC-dA) was o...

Two recombinant phages that contain cardiac muscle actin gene were isolated from a human DNA library and their structures were determined. Restriction analysis indicates that both clones carry the same EcoRI 13-kilobase fragment where the coding sequence is mapped. The cloned DNA hybridized with polyadenylylated RNA from human fibroblasts, which directs the synthesis of cytoplasmic beta- and gamma-actin in vitro. However, sequence determination of the cloned DNA showed that the entire coding sequence perfectly matched the amino acid sequence of cardiac muscle actin. The initiation codon is followed by a cysteine codon that is not found at the amino-terminal site of any actin isoform, suggesting the necessity of post-translational processing for in vivo actin synthesis. There are five introns interrupting exons at codons 41/42, 150, 204, 267, and 327/328. Surprisingly, these intron locations are exactly the same as those of the rat skeletal muscle actin gene but different from those of nonmuscle beta-actin gene. Nucleotide sequences of all exon/intron boundaries agree with the G-T/A-G rule (G-T at the 5' and A-G at the 3' termini of each intron). The 3'-untranslated sequence has no homology to that of nonmuscle beta- or gamma-actin gene, but Southern blot hybridization has shown that this region has considerable homology to that of one of the other actin genes. These results indicate that the recombinant phages, which we have isolated, contain cardiac muscle actin gene and that cardiac muscle actin gene and skeletal muscle actin genes are derived from their ancestor gene at a relatively recent time in evolutionary development.

genomes. We have isolated and characterized TG-elements from different locations in the human genome: from randomly isolated clones, associated with the actin gene family, and linked to another repeated element. The results indicate that the following features are typical of these TGelements: (i) the elements consist of 20 to 60 base pairs of (dT-dG)" * (dC-dA)., (ii) the sequences characterized in our study were not flanked by direct or inverted repeats, (iii) the sequences are interspersed rather than in satellite blocks, (iv) the elements are not usually associated with other repeated elements, and (v) some of the elements are found near coding sequences or in introns. Studies on the conformation of a genomic TG-element in a supercoiled plasmid indicate several distinct properties of the TG-element: (i) it is in the Z-form only at low ionic strength, (ii) S1 nuclease recognizes its Z-form with a marked preference for one of the B-Z junctions, and (iii) the sensitive region extends for 20 base pairs near the B-Z junction. In contrast to the result with the supercoiled plasmid, S1 nuclease failed to recognize the TG-element in minichromosomes.Sequences of alternating purines and pyrimidines such as poly(dG-dC) * poly(dG-dC) and poly(dT-dG) * poly(dC-dA) may assume a left-handed conformation (Z-DNA) under certain conditions (12,22,23,41). Studies with specific antibodies indicate that Z-DNA can be found in acid-fixed chromosomes (2, 20) and supercoiled plasmid and viral DNAs (21, 23), and several interesting biological roles for Z-DNA have been proposed (20,23). Hybridization studies with synthetic poly(dT-dG) * poly(dC-dA) suggest that homologous sequences are abundant and interspersed in the genomes of many, if not all, eucaryotes (8,9). In addition, sequence analysis of various cloned genes occasionally reveals the presence of poly(dT-dG) * poly(dC-dA) in the vicinity of coding regions (e.g., see references 1, 10, 18, 19, 33, 34, and 40). From these data a partial characterization of the structure and organization of this class of poly(dTdG) * poly(dC-dA) sequences has emerged, and on the basis of some of these data it has been proposed that the poly(dTdG) * poly(dC-dA) sequences spread throughout the genome by insertion (26).If the genomic poly(dT-dG) -poly(dC-dA) sequences (TGelements) have a biological function, it is probably because of their conformational peculiarity. The Z-form of poly(dGdC) * poly(dG-dC), another potential Z-DNA sequence, has been extensively studied. The Z-form of poly(dG-dC)-poly(dG-dC) is recognized by specific antibodies (14) and is sensitive to S1 nuclease (31) when in a supercoiled plasmid. The poly(dT-dG) -poly(dC-dA) sequence, when in supercoiled plasmids, also has been shown to be in Z-form (12,22), although its conformational properties are not known in detail. Supercoiled plasmids with genomic TG-elements thus are useful substrates for characterizing the conformational properties of these elements.In this report we present the results of a series of experi-* Corresponding ...