We have developed a cell-free system from Escherichia coli for studying illegitimate recombination between nonhomologous DNA molecules. The recombination is stimulated by oxolinic acid, an inhibitor of DNA gyrase. The stimulation is abolished by coumermycin Al and is not found in extracts of nalidixic acid-resistant (gyrA) mutants. We therefore inferred that DNA gyrase directly participates in illegitimate recombination, at least in the presence of oxolinic acid [Ikeda, H., Moriya, K. & Matsumoto, T. (1981) Cold Spring Harbor Symp. Quant. Biol. 45, 399-408]. The structure of recombinant DNA molecules formed in the presence of oxolinic acid from a cross between phage A and plasmid pBR322 DNAs was analyzed by heteroduplex mapping. Among nine isolates tested, two recombinants were formed by the insertion of the plasmid into the A genome. The seven other recombinants had more complicated genome structures. Insertion of pBR322 was accompanied by a deletion on one of the genomes. In all cases, the end points of deletions coincided with one end of the pBR322 insertion. Recombination sites seemed to be distributed randomly on 'the A and pBR322 genomes. Analysis of nucleotide sequences of the recombination junctions proved that the crossover took place between nonhomologous DNA sequences. A model for DNA gyrase-mediated illegitimate recombination is discussed.Chromosomal rearrangements take place throughout the genomes ofbacteriophages, bacteria, and higher organisms. Tandem duplication appears to be produced by illegitimate recombination between nonhomologous sequences originally present on different DNA molecules (1). Abnormal excision of A prophage from the bacterial chromosome results in the formation of specialized transducing phage such as Agal and Abio (2). These recombinations are generally thought to be recombination between nonhomologous regions or very short homologous regions ofDNA. The illegitimate recombination observed in the Escherichia coli system usually takes place independently of bacterial recA function and viral int, xis, and red functions (1,(3)(4)(5)(6).We have been working to develop a cell-free illegitimate recombination system ofE. coli and have recently shown that an in vitro packaging system consisting of lysates of induced lysogens is capable ofrecombining two heterologous DNA species (7). The packaging mixtures contain a large amount of concatemeric A DNA derived from prophage. This A DNA is a good substrate for illegitimate recombination as well as packaging. When a plasmid DNA is incubated with the packaging mixture, the plasmid recombines with the A DNA and is packaged into a A head, resulting in the formation of recombinant phages. Studies with this system showed that oxolinic acid, an inhibitor of DNA gyrase, stimulates recombination, and the stimulation is abolished by coumermycin Al, another type of gyrase inhibitor. We therefore inferred that DNA gyrase directly participates in this recombination (7). In the present paper, we have studied the structure of

A 3. 1-kilobase Bgl H fragment of Saccharomyces cerevisiae carrying the nuclear gene encoding the mitochondrial polypeptide chain elongation factor (EF) Tu has been cloned on pBR327 to yield a chimeric plasmid pYYB. The identification of the gene designated as tufM was based on the cross-hybridization with the Escherichia coli tufB gene, under low stringency conditions. The complete nucleotide sequence of the yeast tufM gene was established together with its 5'-and 3'-flankdng regions. The sequence contained 1,311 nucleotides coding for a protein of 437 amino acids with a calculated Mr of 47,980. The nucleotide sequence and the deduced amino acid sequence of tufM were 60% and 66% homologous, respectively, to the corresponding sequences of E. coli tufA, when aligned to obtain the maximal homology. Plasmid YRpYB was then constructed by cloning the 2.5-kilobase EcoRI fragment of pYYB carrying tufM into a yeast cloning vector YRp-7. A mRNA hybridizable with tufM was isolated from the total mRNA of S. cerevisiae D13-IA transformed with YRpYB and translated in the reticulocyte lysate. The mRNA could direct the synthesis of a protein with Mr 48,000, which was immunoprecipitated with an anti-E. coli EF-Tu antibody but not with an antibody against yeast cytoplasmic EF-la. The results indicate that the tufM gene is a nuclear gene coding for the yeast mitochondrial EF-Tu.The polypeptide chain elongation factor Tu (EF-Tu) promotes a GTP-dependent binding of an aminoacyl-tRNA to the A site of ribosomes (1). EF-Tu from Escherichia coli consists of a single polypeptide chain with Mr 43,000, and the primary structure comprised of 393 amino acid residues has been determined (2). The protein is encoded by two nearly identical genes on the E. coli chromosome (3), tufA at 73 min and tufB at 89 min (4). Both tufA (5) and tufB (6) have been cloned and their nucleotide sequences determined. The sequences of tufA (7) and tufB (8) are nearly homologous and differ only in 13 positions but the gene products, EF-TuA and EF-TuB, are identical except for the COOH-terminal amino acid (2).In eukaryotes, the counterpart of prokaryotic EF-Tu, designated as EF-la, has been purified from various sources, including pig liver (9), rabbit reticulocytes (10), Artemia salina (11), wheat germ (12), and yeast (13), and was shown to consist of a single polypeptide chain, Mr 47,000-53,000. The partial amino acid sequence of EF-la from rabbit reticulocytes (14) and A. salina (15) was determined, and sequence conservation between A. salina EF-la and E. coli EF-Tu has been reported (15).In addition to EF-la, which functions in the cytoplasmic fraction in conjunction with 80S ribosomes, eukaryotic cells possess mitochondrial EF-Tu (designated as mEF-Tu) that functions in the mitochondrial translational apparatus (16,17). Translational factors as well as ribosomal proteins in the mitochondria are encoded by nuclear genes, synthesized in cytoplasmic fractions, and transported into the mitochondria (18). The translational machineries of mitochondria have...

The TaqI t allele of the VDR gene may be a risk factor for severe stone disease and recurrent stones.

Previous studies in hereditary and sporadic prostate cancer have indicated the existence of a tumor suppressor gene in chromosomal region 19p13. The BRG1 gene in this region is one of the possible candidates, based on both the frequency of inactivating mutations in human cancer cell lines, including the prostate cancer cell line DU145, and its functional properties. To our knowledge, no studies have been done to evaluate possible involvement of the BRG1 gene in clinical prostate cancer. To accomplish this, we carried out a complete mutation analysis of all 35 BRG1 exons in tumor and constitutional DNA samples from 21 prostate cancer patients. We report the absence of somatic mutations in the panel of samples employed, but the existence of five germline single nucleotide polymorphisms (SNPs) in CpG islands of the BRG1 gene, among them, three novel ones. In conclusion, the study excludes the presence of common BRG1 mutations in prostate cancer.

We have previously shown that DNA gyrase of Escherichia coli can promote recombination between heterologous DNAs in a cell-free system (Ikeda et al. 1982). In the present paper, we have studied the nucleotide sequences of several recombination junctions of lambda-pBR322 recombinants and found that there were not more than three-basepair homologies between the parental DNAs in six combinations of the lambda and pBR322 recombination sites. Based on this and previous results, we concluded that homology was not required for the DNA gyrase-mediated recombination. Furthermore, the structures of the recombinant DNAs we have analyzed suggest the occurrence of multiple cross-overs in our in vitro system.