Molecules involved in cell–cell adhesion during development

A peculiarity of nitrosamines is the high degree of cell and organ specificity in inducing tumors. There is substantial evidence that the initiation of the carcinogenesis process by carcinogens of this group is linked to the metabolic competence of the target tissue or cell to convert these carcinogens into mutagenic metabolites and to the binding of those metabolites to cellular DNA. Alkylation occurs in the DNA at the N-1, N-3, and N-7 positions of adenine; the N-3, N-7, and O⁶ of guanine; the N-3, and O² of cytosine; and the N-3, O⁴, and O² of thymine; and the phosphate groups. The initial proportion of each DNA adduct depends upon the alkylating agent used. The various DNA adducts are lost to a variable extent from DNA in vivo by spontaneous release of bases and Or by specific DNA repair processes. Studies conducted in vitro and in vivo indicate that alkylation at the oxygen atoms of DNA bases is more critical than alkylation at other positions in the mutagenesis and carcinogenesis induced by N-nitroso compounds. In particular, tissues in which tumors occur more frequently after a pulse dose of nitrosamine are those in which O⁶-alkylguanine persists longest in DNA, presumably resulting in an increased probability that a miscoding event (mutation) will take place during DNA synthesis. The more rapid removal of O⁶-methylguanine from the DNA of liver (as compared with cxtrahepatic tissues) of rats has been associated with the absence of tumor production in this organ by a single dose of dimethylnitrosamine; however, a significant incidence of liver tumors is observed if the same dose is given 24 hr after partial hepatectomy, and tumors arc induced by such a dose of dimethyl-nitrosamine in the liver of hamsters, which has a low capacity to remove O⁶-methylguanine from its DNA. These data also indicate that the rate of disappearance of 7-methylguanine from the liver or cxtrahepatic tissues is independent of the dose of dimethylnitrosamine; whereas O⁶-methylguanine is lost from DNA more rapidly after a low dose of this nitrosamine. It has been shown that in liver the removal of O⁶-methylguanine, but not of other DNA adducts, from DNA can be affected by pretreating the animals with N-nitroso compounds. The modulation of DNA repair processes observed after a single dose and after chronic treatment with nitrosamines is discussed in relation to the tissue-specific carcinogenic effect of this group of carcinogens.     

Comparaisons des séquences d'ADN hautement répétées chez l'homme et diverses espèces de primates

Highly repeated DNA sequences from man, five other primate species and rat were compared using five restriction endonucleases. Calculations of a similarity index based on the mobility of various bands indicate that man, chimpanzee and baboons are very similar. The sequences of the genomes studied have apparently been reorganized during primate evolution.     

Simple and efficient cell disruption of extremely small quantities of mycelium of phytopathogenic mycotoxin-producing moulds for quantitative extraction of genomic DNA

DNA was efficiently and quantitatively isolated from extremely small quantities of mycelia (0.1–10 mg) of different phytopathogenic moulds by grinding freeze-dried mycelia with glass beads and then using a commercial DNA extraction kit. The efficiency of disruption of the mycelia and the quantitative DNA extraction was proved by microscopy and the quantification of isolated DNA by real time PCR. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005 Financial support: German Research Foundation (DFG grant Pr 708/2). J.M. thanks the Cusanuswerk for a doctoral scholarship     

Absence of repetitive DNA sequences associated with sex chromosomes in natural populations of the Trinidad guppy (Poecilia reticulata)

The genus Poecilia has been widely studied as a model for the evolution of sex chromosomes. In the course of molecular studies on population genetic structure and sexual selection in the Trinidad guppy, we examined our preparations for male-linked, repetitive DNA polymorphisms. We have not obtained any evidence of male-specific polymorphisms, in contrast to an earlier study. Our results have significant implications for theories on the evolution of sex chromosomes.Correspondence to: F. Breden     

Efficiency in cloning and sequencing using the single-stranded bacteriophage M13

A number of approaches to sequence DNA by the chain termination method are based on cloning into M13 phage vectors and the use of a universal primer. In this paper we investigate some of the factors which influence the speed and efficiency of these approaches. A modification of the template preparation, sequencing reaction, and gel system was used to obtain more reliable and clearer ladder gels. Redundancy and deficiency of shotgun DNA sequencing were reduced by a mapping technique and the use of synthetic primers. The mapping and cloning technique was used to organize the data entry so that the computer time necessary to reconstruct the sequence out of overlaps was reduced.     

A transient species of poly(A)+ RNA detected by a myosin heavy chain cDNA probe in muscle cell culture during terminal differentiation

Primary cell cultures were prepared from breast muscles of 11 day 4 hour-embryonic chicks. Cytoplasmic RNAs were isolated from the cultured cells at various time intervals from day 3 to day 8. A [P32] DNA probe complementary to messenger RNA of myosin heavy chain was used to hybridize with the RNAs after gel electrophoresis. A transient species of polyadenylated RNA with a decreased mobility in electrophoresis was detected during a period of time when contractions of syncytial fibers were first observed.     

Isolation of the gene encoding pilin of Bacteroides nodosus (strain 198), the causal organism of ovine footrot

The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin-producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence.     

Analysis by electron microscopy of the variable segment in the maxi-circle of kinetoplast DNA from Trypanosoma brucei

The 20.5-kbp maxi-circle from the kinetoplast DNA of Trypanosoma brucei contains a 5-kbp segment which is not cut by most restriction endonucleases and which varies in size in closely-related trypanosome strains (Borst, P., Fase-Fowler, F., Hoeijmakers, J.H.J. and Frasch, A.C.C. (1980) Biochim. Biophys. Acta 610, 197–210). We have now analysed partial denaturation maps of the linearized maxi-circles by electron microscopy and find that the variable segment is not more AT-rich than the remainder of the maxi-circle. Early denaturation begins at two separate regions of the maxi-circle outside the variable region and one of these corresponds with the position of the gene for the large (12 S) ribosomal RNA. Denaturation-renaturation of maxi-circles leads to the formation of partially mismatched duplexes that look like underwound loops in electron micrographs. These loops are only found in the variable region and they vary in size and appearance. Under our renaturation conditions single-stranded maxi-circle DNA is devoid of secondary structure and this suggests that the underwound loops arise by misalignment of straight tandem repeats in the DNA. We have also analysed heteroduplexes between maxi-circles from two closely related T. brucei strains that differ by 1 kbp in the size of their variable segment. Most molecules had no underwound loops and contained mismatched regions in the variable segment only. The appearance of these regions is diverse, varying from fully duplex with two single-stranded loops to molecules with a heterogeneous array of smaller loops. The total size of single-stranded DNA in the heteroduplexes may be as high as 1.2 μm, i.e., a factor 4 higher than the size difference between the heteroduplex partners. We conclude that the variable region consists of imperfect tandem repeats of a sequence that evolves rapidly. This region might contain the origin of maxi-circle replication.     

Disassembly of Escherichia coli RecA E38K/��C17 Nucleoprotein Filaments Is Required to Complete DNA Strand Exchange

Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/ΔC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/ΔC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/ΔC17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.