A meiosis-specific protein kinase, Ime2, is required for the correct timing of DNA replication and for spore formation in yeast meiosis

 In this report we study the regulation of premeiotic DNA synthesis in Saccharomyces cerevisiae. DNA replication was monitored by fluorescence-activated cell sorting analysis and by analyzing the pattern of expression of the DNA polymerase α-primase complex. Wild-type cells and cells lacking one of the two principal regulators of meiosis, Ime1 and Ime2, were compared. We show that premeiotic DNA synthesis does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. At late meiotic times, ime2Δ diploids exhibit an additional round of DNA synthesis. Furthermore, we show that in wild-type cells the B-subunit of DNA polymerase α is phosphorylated during premeiotic DNA synthesis, a phenomenon that has previously been reported for the mitotic cell cycle. Moreover, the catalytic subunit and the B-subunit of DNA polymerase α are specifically degraded during spore formation. Phosphorylation of the B-subunit does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. In addition, we show that Ime2 is not absolutely required for commitment to meiotic recombination, spindle formation and nuclear division, although it is required for spore formation. Received: 20 February 1996 / Accepted: 7 June 1996     

Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations

Summary Postreplication repair of nuclear DNA was examined in an excision defective haploid strain of yeast lacking mitochondrial DNA (ral ⁰). The size of the DNA synthesized in cells exposed to various fluences of ultraviolet light (UV) corresponds approximately to the average interdimer distance in the parental DNA. Upon further incubation of cells following exposure to 2.5 J/m², the DNA increases in size; by 4 h, it corresponds to DNA from uniformly labeled cells. The alkaline sucrose sedimentation pattern of DNA pulse labeled at various times after UV irradiation, for up to 4 h, does not change substantially, indicating that dimers continue to block DNA replication. A significant amount of postreplication repair requires de novo protein synthesis, as determined by its inhibition by cycloheximide. The rad6 mutant does not carry out postreplication repair, the rad18 and rad52 mutants show great inhibition while the rev3 mutation does not affect postreplication repair. Both recombinational and nonrecombinational repair mechanisms may function in postreplication repair and most of postreplication repair is error free.     

The fate of the bacterial chromosome in P22-infected cells of Salmonella typhimurium

Summary Evidence is presented that in cells of Salmonella typhimurium infected with phage P22 there is no conversion of bacterial DNA material into phage DNA material. It has been shown also that infection with P22 does not cause measurable breakdown of the bacterial chromosome to phage-sized or smaller DNA fragments.     

The effect of single-stranded DNA binding proteins on template/primer-independent DNA synthesis in the presence of nicking endonuclease Nt.BspD6I

Nt.BspD6I nicking endonuclease stimulates template/primer-independent DNA synthesis by Bst DNA polymerase. Template/primer-independent DNA synthesis may be one of the reasons for the formation of nonspecific products in certain DNA amplification reactions, especially those involving nicking endonucleases. Expansion of the range of DNA amplification procedures performed in the presence of nicking endonucleases makes the search for template/primer-independent DNA synthesis inhibitors highly relevant. The present work has shown that a single-strand DNA binding protein from E. coli does not affect template/primer-independent DNA synthesis regardless of the presence or absence of Nt.BspD6I. A single-stranded DNA-binding protein coded by gene 32 from bacteriophage T4 completely inhibits template/primer-independent DNA synthesis in the absence of nicking endonuclease. If nicking endonuclease is present, the protein does not suppress the synthesis of the specific product but causes a significant decrease of the amount of template/primer-independent DNA synthesis products.     

The occurrence and role of DNA synthesis during meiosis in wheat and rye

The incorporation of ³H-thymidine into the DNA of rye meiocytes at zygotene, pachytene-diplotene and metaphase I to telophase II stages has been studied. Low levels of ³H were found in highly purified DNA from meiocytes at all these stages, though there was more in the DNA from pachytene-diplotene meiocytes, and it is highly likely that the zygotene groups of anthers contained a proportion at pachytene. The buoyant density distributions of the labelled DNA from zygotene and pachytene-diplotene cells were indistinguishable, in contrast to the situation in Lilium, the only other example studied so far.The DNA synthesis inhibitor 2′-deoxyadenosine halted meiotic development of anthers in culture only at late zygotene and pachytene. It did not inhibit development at early zygotene, prevent chromosome pairing as judged by light microscopy or cause extensive chromosome fragmentation during zygotene as in Lilium. These results indicate that extensive synthesis of DNA does not occur at zygotene in cereals and does not suggest that zygotene DNA synthesis is a prerequisite for chromosome pairing as in Lilium.     

Specific inactivation of heterospecific transforming DNA by a factor derived from Streptococcus sanguis lysates.

Summary A heat-sensitive factor obtained from lysates of competent Streptococcus sanguis cells reacts specifically with native DNA of heterospecific (S. pneumoniae or calf thymus) origin. In vitro it does not alter the double or single strand length of the DNA, nor does it affect uptake of the DNA by competent S. sanguis or S. pneumoniae cells in DNase I-resistant form. Following uptake, however, DNA previously exposed to the factor loses over 90% of its biological activity. Reaction of heterospecific DNA with the factor is cometitive, suggesting a competition for binding to the factor. Heating treated DNA prior to its reaction with recipient cells, apparently by irreversibly dissociating the factor, restores to the DNA its original potential transforming activity. Specific activity of the factor can be increased in cells grown under certain conditions; this increase is blocked by erythromycin.     

An unstable donor-recipient DNA complex in transformation of Bacillus subtilis.

Summary In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 5¹, 8-trimethylpsoralen in conjuction with long-wave ultra violet light irradiation. This indicates that base-pairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation.     

A novel role for fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells

Fanconi Anemia (FA) is a cancer-susceptibility syndrome characterized by cellular sensitivity to DNA inter-strand cross-link (ICL)-inducing agents. The Fanconia Anemia D2 (FANCD2) protein is implicated in repair of various forms of DNA damage including ICLs. Studies with replicating extracts from Xenopus eggs indicate a role for FANCD2 in processing and repair of DNA replication-associated double stranded breaks (DSB). We have investigated the role of FANCD2 in cell cycle progression of cultured human cells. Similar to Xenopus cell-free extracts, we show that chromatin association of FANCD2 in human cells is coupled to ongoing DNA replication. siRNA depletion experiments demonstrate that FANCD2 is necessary for efficient DNA synthesis. However, in contrast with Xenopus extracts, FANCD2-deficiency does not elicit a DNA damage response, and does not affect the elongation phase of DNA synthesis, suggesting that FANCD2 is dispensable for repair of replication-associated DNA damage. Using synchronized cultures of primary untransformed human dermal fibroblasts we demonstrate that FANCD2 is necessary for efficient initiation of DNA synthesis. Taken together, our results suggest a novel role for the FA pathway in regulation of DNA synthesis and cell cycle progression. Inefficient DNA replication may contribute to the genome instability and cancer-propensity of FA patients.     

Evaluation of possible horizontal gene transfer from transgenic plants to the soil bacterium Acinetobacter calcoaceticus BD413

 The use of genetically engineered crop plants has raised concerns about the transfer of their engineered DNA to indigenous microbes in soil. We have evaluated possible horizontal gene transfer from transgenic plants by natural transformation to the soil bacterium Acinetobacter calcoaceticus BD413. The transformation frequencies with DNA from two sources of transgenic plant DNA and different forms of plasmid DNA with an inserted kanamycin resistance gene, nptII, were measured. Clear effects of homology were seen on transformation frequencies, and no transformants were ever detected after using transgenic plant DNA. This implied a transformation frequency of less than 10^-13 (transformants per recipient) under optimised conditions, which is expected to drop even further to a minimum of 10^-16 due to soil conditions and a lowered concentration of DNA available to cells. Previous studies have shown that chromosomal DNA released to soil is only available to A. calcoaceticus for limited period of time and that A. calcoaceticus does not maintain detectable competence in soil. Taken together, these results suggest that A. calcoaceticus does not take up non-homologous plant DNA at appreciable frequencies under natural conditions. Received: 1 November 1996 / Accepted: 18 April 1997     

DNA helicase mutants of bacteriophage T4 that are defective in DNA recombination

We previously isolated a plasmid-borne, recombination-deficient mutant derivative of the bacteriophage T4 DNA helicase gene 41. We have now transferred this 41rrh1 mutation into the phage genome in order to characterize its mutational effects further. The mutation impairs a recombination pathway that is distinct from the pathway involving uvsX, which is essential for strand transfer, and it also eliminates most homologous recombination between a plasmid and the T4 genome. Although 41rrh1 does not affect T4 DNA replication from some origins, it does inactivate plasmid replication that is dependent on ori(uvsY) and ori(34), as well as recombination-dependent DNA replication. Combination of 41rrh1 with some uvsX alleles is lethal. Based on these results, we propose that gene 41 contributes to DNA recombination through its role in DNA replication. Received: 3 February 1999 / Accepted: 20 July 1999     

A new type of IS1-mediated deletion

Summary Genetical tests and DNA sequence analysis revealed that the mechanism of formation of IS1-induced type I and type II deletions differs.IS1-mediated type II deletions occur at the termini of the integrated element and do not remove the element. This process is independent of the cellular recA system and does not involve DNA sequence homology.Conversely, the formation of IS1-induced type I deletions differs substantially. They require recA gene product, small DNA sequence duplications and a topological arrangement of the DNA molecule to allow alignment of duplications.     

Studies on DNA Topoisomerase activity during in vitro chromatin assembly

The in vitro assembly of chromatin, promoted by the Xenopus cell-free extract (S-150), can be inhibited by oxolinic acid and to a lesser extent by nalidixic acid. Both of these antibiotics have been shown to block the activity of the specialized type 11 Topoisomerase, bacterial DNA Gyrase. Oxolinic acid induces a DNA cleavage by Micrococcal Nuclease at specific sequences in the multiple cloning vector pGEM-4. Nalidixic acid does not inhibit DNA supercoiling, but does diminish the extent of chromatin formation achieved by the S-150 on circular DNA templates. The Topoisomerase I inhibitor, berenil, does not inhibit extensive chromatin assembly, although it aloes diminish the level of supercoiling. Taken together, these results suggest that both topoisomerases play a role in the assembly process. Topoisomerase I may catalyze both the introduction of unconstrained supercoils into relaxed DNA and the formation of monosomes, while Topoisomerase 11 may promote extended chromatin assembly.     

The evolution of a family of short interspersed repeats in primate DNA

Summary Human DNA contains 300 nucleotide interspersed repeated sequences which mostly belong to a single family of sequences called the Alu family. This work examines the evolution of this family of sequences in primates. Bonnet monkey (Macaque radiata) DNA contains a predominant family of 300 nucleotide repeats which has nearly the same restriction map as the human Alu family and which hybridizes to human Alu family repeats under Southern blotting conditions. Prosimian (Galago crassicaudatus pangeniesis) DNA also contains a prominent group of 300 nucleotide long repeated sequences which does not have the same restriction sites as the human Alu family but which does hybridize to the human Alu family under reduced stringency conditions.     

Semi-in vitro Repair of DNA in Germinating Spores of Bacillus subtilis Irradiated with γ-Ray

DNA repair synthesis was studied in germinating spores of Bacillus subtilis made permeable to deoxyribonucleoside triphosphates by treatment with Brij 58. The synthesis is dependent on the presence of all four deoxyribonucleoside triphosphates, but does not require adenosine triphosphate. Repair synthesis in the γ-ray irradiated and Brij 58 treated germinating spores was observed in wild type strain 168Tt, but not in DNA polymerase I-deficient mutant strain D22. Furthermore, the single-strand breaks of DNA in the germinating spores of strain 168Tt induced by γ -ray irradiation were rejoined during postirradiation incubation in the presence of four deoxyribonucleoside triphosphates, nicotinamide adenine dinucleotide and magnesium ion. In the case of a mutant D22, the γ-ray induced DNA single-strand breaks were not rejoined.     

Characterization of a dinoflagellate (Amphidinium carterae) DNA

The DNA of the photosynthetic Dinoflagellate Amphidinium carterae shows, by certain physico-chemical criteria two major components, a light satellite and a permanent DNA-RNA hybrid whose presence explains the unusual behaviour of Amphidinium DNA according to classical methods of analysis. The frequent presence of a slow-growing accompanying bacterium does not seem to interfere with any of these findings.     

Recombination and genome size

Summary Within complements the chiasma frequency per chromosome, which directly reflects the amount of recombination, is generally closely correlated with chromosome length, i.e. the chromosomal DNA content. The correlation does not apply when comparisons are made between the complements of different species. Analyses of results from three Angiosperm genera show a progressive decrease in the chiasma frequency per picogram of DNA with increase in nuclear DNA amount.     

Asymmetric repair of bacteriophage T7 heteroduplex DNA

Summary Heteroduplex DNA molecules were prepared in vitro using one strand of DNA carrying a point mutation and one strand of the corresponding wild-type DNA. The heteroduplex DNA was transfected into competent bacteria and the progeny genotypes in the resulting infective centers were determined. From the results were conclude that about 80% of all transfected DNA molecules are repaired before DNA replication starts. This fraction of repaired DNA is independent of the location of the mismatched nucleotide pair. However, mismatch correction occurs preferentially on the H strand of the heteroduplex DNA.The repair does not depend on a known phage coded function but requires the active bacterial genes mut U, mut H, mut S and probably mut L.     

A rapid spectrofluorimetric method for the determination of the degree of synchronism inSaccharomyces cerevisiae

A fluorescence technique that allows direct measurement of DNA and synchrony levels inSaccharomyces cerevisiae cell cultures is reported. The spectrofluorimetric estimation of DNA with 4,6-diamidino-2-phenylindole (AT-dye) does not require prior extraction, is highly stable, and requires small quantities of cells.     

GATA repeats in the genome of Asellus aquaticus (Crustacea,Isopoda)

A 500 bp fragment of Drosophila genomic DNA containing 37 copies of the tetranucleotide GATA was used to probe, by Southern DNA blotting and in situ hybridization, two natural populations of the isopod crustacean Asellus aquaticus collected from the Sarno and Tiber rivers. This species does not have a recognizable sex chromosome pair. In a number of males from the Sarno population chromomycin A₃ staining reveals a heteromorphic chromosome pair. The heterochromosome has two blocks of heterochromatin. After digestion of genomic DNA with six restriction endonucleases and hybridization with the GATA probe, the two populations exhibit different fragment length patterns. No sex-linked pattern was observed in either population. In situ hybridization to chromosomes of males and females from the Sarno population does not reveal any sex-specific pattern of labelling and indicates a scattered distribution of GATA sequences on most chromosomes with some areas of preferential concentration. The heterochromatic arcas of the male heterochromosome are not labelled.by E.R. Schmidt