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...

A recombinant phage that carries the cytoplasmic .8-actin gene was isolated from a human DNA library.The nucleotide sequence of this gene was determined. The amino acid sequence deduced from the nucleotide sequence matches perfectly that of f8-actin from human fibroblasts. The gene contains five introns. A large intron was found in the 5' untranslated region six nucleotides upstream from the ATG initiation codon. Four introns were found within the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. In contrast to the human cardiac muscle actin gene, the aorta-type smooth muscle actin gene, and the stomach-type smooth muscle actin gene, the .-actin gene lacks

A31-714, a subclone isolated from clone A31 of the BALBI3T3 line showed a high degree of contact inhibition and an extremely low incidence of spontaneous transformation, Treatment of this subclone with 4-nitroquinoline-I-oxide, 3-methylcholanthrene, benzo (alpyrene or N-methvl

Recent analyses of the three-dimensional structure of synthetic DNA molecules has revealed the existence of a left-handed double-helical conformation of DNA, called Z-DNA. The Z-DNA structure was first observed in molecules having alternating guanine and cytosine bases, but has now also been shown for molecules of sequence poly(dT-dG) . poly(dC-dA) (refs 4-7). If Z-DNA occurs naturally then it might have quite different reactivities with molecules such as proteins or carcinogens from right-handed B-DNA. The interconversion of sequences between B and Z forms, under the influence, for example, of DNA binding proteins or chemical modification, may be important in regulating DNA function. So far, little data have been published on the occurrence of potential Z-DNA forming sequences in eukaryotic DNA. Here we report the presence of a sequence of 50 alternating dT and dG residues within one of the introns of a human cardiac muscle actin gene. Also, using a probe specific for poly(dT-dG) sequences, we have also found that potential Z-DNA forming sequences are highly repeated in the human genome.

Recombinant phages that carry the human smooth muscle (enteric type) -y-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle -y-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.

A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p6(a-Cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3' end in the last coding exon, which probably resulted in a deletion and an altered C' terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor.

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.

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