DNS-Replikationsmuster der Speicheldrüsen-Chromosomen von Chironomiden

The pattern of DNA-synthesis of the salivary gland chromosomes of Chironomus thummi thummi, Ch. th. piger, Ch. annularius, Ch. plumosus and Ch. melanotus was studied using H³-thymidine-autoradiography. Contrary to the previous conception the bands of the salivary gland chromosomes of Chironomus do not begin replication simultaneously. H³-thymidine incorporation in bands of high DNA content begins later than in bands with a lesser amount of DNA. This difference in time is very small in bands outside the kinetochore regions and not comparable to the asynchrony in replication of typical heterochromatin in the salivary gland chromosomes of Chironomus melanotus. Differences in the amount of DNA in homologous bands do not affect the onset of replication. — Bands of high DNA content are replicating during a longer time than those having less DNA. However, certain chromosome regions behave differently. In these regions bands of very low DNA content are synthesizing DNA during the whole replication cycle. Since no excessive increase of DNA could be observed in these regions it is supposed that in addition to the duplication of structural DNA an extra DNA is synthesized which disappears immediately from the chromosome. — At the end of the replication cycle in the salivary gland nuclei of the hybrid Chironomus th. thummi X Ch. th. piger a labeling pattern is found in the chromosomes of Ch. th. thummi which differs from that in the parental subspecies Ch. th. thummi.     

Sgs1 and Exo1 suppress targeted chromosome duplication during ends-in and ends-out gene targeting

Gene targeting is extremely efficient in the yeast Saccharomyces cerevisiae. It is performed by transformation with a linear, non-replicative DNA fragment carrying a selectable marker and containing ends homologous to the particular locus in a genome. However, even in S. cerevisiae, transformation can result in unwanted (aberrant) integration events, the frequency and spectra of which are quite different for ends-out and ends-in transformation assays. It has been observed that gene replacement (ends-out gene targeting) can result in illegitimate integration, integration of the transforming DNA fragment next to the target sequence and duplication of a targeted chromosome. By contrast, plasmid integration (ends-in gene targeting) is often associated with multiple targeted integration events but illegitimate integration is extremely rare and a targeted chromosome duplication has not been reported. Here we systematically investigated the influence of design of the ends-out assay on the success of targeted genetic modification. We have determined transformation efficiency, fidelity of gene targeting and spectra of all aberrant events in several ends-out gene targeting assays designed to insert, delete or replace a particular sequence in the targeted region of the yeast genome. Furthermore, we have demonstrated for the first time that targeted chromosome duplications occur even during ends-in gene targeting. Most importantly, the whole chromosome duplication is POL32 dependent pointing to break-induced replication (BIR) as the underlying mechanism. Moreover, the occurrence of duplication of the targeted chromosome was strikingly increased in the exo1Δ sgs1Δ double mutant but not in the respective single mutants demonstrating that the Exo1 and Sgs1 proteins independently suppress whole chromosome duplication during gene targeting.     

Evidence for sequence preferences in the intercalative binding of ethidium bromide to dinucleoside monophosphates.

The rate of production of tandem duplications in phage λ has been measured in the presence and absence of known recombination systems. Two deletion phages have been used: tdel33, a deletion derivative of a φ80-λ hybrid phage, and λb221, which carries a large deletion of the central portion of the λ chromosome. Both phages are int⁻, and tdel33 is also red⁻, by virtue of their deletions. Stocks of these phages can be prepared free of long tandem duplication derivatives by CsCl density gradient purification. After a single cycle of lytic growth, lysates from these purified phage stocks contain tandem duplications at a frequency of 10⁻³ in the case of tdel33 and 10⁻⁵ in the case of λb221. These frequencies are unaffected by the presence of mutations in the host Rec system or the phage Red system. To investigate the difference in duplication frequency between tdel33 and λb221, the phages were grown in mixed infection. The result indicates that a trans-active product of tdel33 is responsible for its high frequency of duplication production.Tandem duplications have been detected by banding the phage lysates in CsCl density gradients. Long DNA addition mutants can be detected in this way if they arise with a frequency of at least 10⁻⁵ and if the duplication length is at least 0.14 λ lengths. To accomplish this it is necessary to distinguish them from contaminating parental phage and from dense phages with aberrant structures which arise at roughly comparable frequencies. The former can be done by rebanding and the latter by growth and rebanding. To distinguish these types we have also made use of a new mutant of Escherichia coli which does not plate λ deletion phages. All of the DNA addition mutants we have detected in this way are tandem duplications; evidently mutants with long insertions arise more rarely.     

Multiple Pathways of Duplication Formation with and Without Recombination (RecA) in Salmonella enterica

Duplications are often attributed to “unequal recombination” between separated, directly repeated sequence elements (>100 bp), events that leave a recombinant element at the duplication junction. However, in the bacterial chromosome, duplications form at high rates (10⁻³–10⁻⁵/cell/division) even without recombination (RecA). Here we describe 1800 spontaneous lac duplications trapped nonselectively on the low-copy F′₁₂₈ plasmid, where lac is flanked by direct repeats of the transposable element IS3 (1258 bp) and by numerous quasipalindromic REP elements (30 bp). Duplications form at a high rate (10⁻⁴/cell/division) that is reduced only about 11-fold in the absence of RecA. With and without RecA, most duplications arise by recombination between IS3 elements (97%). Formation of these duplications is stimulated by IS3 transposase (Tnp) and plasmid transfer functions (TraI). Three duplication pathways are proposed. First, plasmid dimers form at a high rate stimulated by RecA and are then modified by deletions between IS3 elements (resolution) that leave a monomeric plasmid with an IS3-flanked lac duplication. Second, without RecA, duplications occur by single-strand annealing of DNA ends generated in different sister chromosomes after transposase nicks DNA near participating IS3 elements. The absence of RecA may stimulate annealing by allowing chromosome breaks to persist. Third, a minority of lac duplications (3%) have short (0–36 bp) junction sequences (SJ), some of which are located within REP elements. These duplication types form without RecA, Tnp, or Tra by a pathway in which the palindromic junctions of a tandem inversion duplication (TID) may stimulate deletions that leave the final duplication.     

VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : III. Effect of Cycloheximide on the Recovery from B12-Induced Unbalanced Growth

When cycloheximide is added to (B12)-deficient cultures before or after replenishment of the cells with B₁₂, reversion of these cells is inhibited. This inhibition is not caused by interference of the inhibitor in the uptake of B₁₂ as measured by division kinetics. Cycloheximide does not inhibit the initial increase in the rate of DNA synthesis caused by B₁₂ replenishment, but within 30–45 min the rate decreases and DNA synthesis ceases. Cycloheximide added to replenished deficient cells after completion of DNA duplication inhibits cell division. The total cellular protein and RNA in replenished cells treated with cycloheximide does not change. B₁₂ added to deficient cells does not stimulate the incorporation of [¹⁴C]leucine into protein during resumption and completion of DNA duplication. However, there is a large increase in [¹⁴C]leucine incorporation into the protein of these cells soon after completion of DNA duplication and before resumption of cell division. The addition of cycloheximide to B₁₂-replenished or to nonreplenished deficient cells rapidly inhibits the incorporation. We suggest that the addition of B₁₂ accelerates the rate of DNA synthesis in the deficient cells and that possibly no new protein synthesis is required except for mitosis. However, protein synthesis is needed for continuous DNA synthesis.     

Bacteriophage lambda derivatives carrying two copies of the cohesive end site

A spontaneously arising tandem duplication derivative of bacteriophage lambda has been isolated, which carries two copies of the site where the cohesive ends are formed (designated cos). Its structure has been determined by electron microscopy of DNA heteroduplexes. These heteroduplexes reveal that the duplication is usually, but not always, carried on the left end of the chromosome. A second duplication phage having two copies of cos, constructed by Feiss &; Campbell (1974), has also been studied by electron microscopy and is found to have a similar property.Unlike most tandem duplication derivatives of phage λ, the mutant studied here is not stable during growth in the absence of generalized recombination, but segregates both the triplication and the parental phage. This verifies that both cos sites are functional. The triplication does not arise as a result of end-to-end aggregation of phage chromosomes or site-specific recombination catalyzed by the chromosome maturation system at cos. It must therefore result from the cutting of mature ι chromosomes from concatemeric replication intermediates. The pattern of cutting observed shows that the λ cohesive ends are not created by a free nuclease acting on unpackaged DNA. The cutting appears to be influenced by the amount of DNA previously packaged into a phage head. A model for λ packaging is presented which explains the results.The duplication phage of Feiss &; Campbell (1974) carries a novel addition containing self-complementary sequences.     

Extent and location of DNA synthesis associated with a class of Rec-mediated recombinants of bacteriophage lambda

The extent and location of DNA synthesis associated with Rec recombination of a lambda phage mutant has been determined approximately for recombinants arising under conditions that restrict DNA duplication. The mutant bio1 contains a substitution in its DNA, and nearly all phage maturing under these conditions have undergone a recombination event within a short region in or near the inserted DNA. Density labeled phage bio1 were used to prepare a lysate under these conditions and the extent of new DNA synthesis was determined by analyzing the density of the progeny phage. On the average, about 6% of the phage chromosome was resynthesized in such a cross.DNA was extracted from bio1 phage crossed under similar conditions in the presence of ³²PO₄. The position of incorporated ³²PO₄ was determined by cleaving the DNA with EcoRI restriction endonuclease and resolving the resulting fragments by electrophoresis on agarose gels. The fragment found to have the most newly synthesized DNA and the highest average amount of synthesis per nucleotide contains the bio1 insertion near its left end and the “hot spot” for Rec-mediated recombination near its center. It appears that in these crosses recombination-associated DNA synthesis is localized about the region of the Rec-mediated recombination event.     

Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Endoreplication Cycles

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM–chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM–chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM–chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.     

Induction and Transmission of a Merodiploid Condition near the Terminal Area of the Chromosome of BACILLUS SUBTILIS

A small fraction (about 0.5%) of the transformants for a particular marker of B. subtilis (ilvA4; most probably a deletion) were found to be relatively unstable merodiploids. They possess a redundancy of the metB–ilvA chromosome segment. When their DNA is used as donor in transformation a merodiploid condition for the whole of this segment is created in all ilvA4⁺ transformants. For several of the duplicated loci both copies often are of recipient strain origin. Markers originally belonging to different copies of the diploidized region can be contransferred in PBS1-mediated transduction. The data are well in agreement with the hypothesis that the merodiploids carry a tandem duplication. An alternative hypothesis which does not call for integration of the exogenote within the recipient chromosome was also considered. Models are proposed for interpreting the segregation of the merodiploids, the transmission of the diploid state and its generation during transformation of the ilvA4 marker by wild-type DNA.     

Karyotype,genome size,and in vitro chromosome doubling of Pfaffia glomerata (Spreng.) Pedersen

Pfaffia glomerata (Spreng.) Pedersen, known worldwide as Brazilian ginseng, has an important commercial value due to its pharmaceutical properties. In addition to the newly described karyological traits and the first estimation of DNA content, this study reports a protocol for the successful induction of tetraploidy. Natural diploid individuals (2n = 34) showed a symmetric karyotype, centromeric DAPI⁺ bands, one chromosome pair with a CMA⁺ band and 45S rDNA site and another with one 5S rDNA site. To induce chromosome duplication, small nodal buds were cultured in semi-solid MS-based medium with 2.22 μM BA, 2.69 μM NAA, and colchicine or oryzalin at 10, 15, 20, 25, and 30 μM for 1 or 2 weeks before being transferred to MS basal medium. The results showed that colchicine induced tetraploid plants, mainly after 1 week of exposure, whereas oryzalin treatment induced only mixoploid plants. The tetraploid plants exhibited twice the chromosome number and DNA content and twice the number of chromosome markers observed for the diploids. Chromosome duplication reduced the dry mass of the stems and roots of the polyploid plants compared to the diploids, and the stomatal density was also reduced on the abaxial and adaxial leaf surfaces of the polyploids. Additionally, the production of β-ecdysone was 50 % higher in the tetraploids than in the diploids. Thus, chromosome doubling showed that is possible to increase the content of β-ecdysone, highlighting the considerable potential of this technique to produce new cultivars with high commercial value.     

Asynchronous Duplication of Chromosomes in Cultured Cells of Chinese Hamster

Chromosome duplication (DNA synthesis) was studied in cultured cells of Chinese hamsters by means of autoradiography following thymidine-H³ incorporation. The technique used was to expose an asynchronously dividing population of rapidly growing cells for a 10 minute interval to a medium with thymidine-H³. Cells were then transferred to a medium with excess unlabeled thymidine. The population was sampled at intervals thereafter and studies made of the frequency of labeled interphases and division figures, and the patterns of labeling of specific chromosomes. The average generation time during these experiments was about 14 hours. DNA synthesis occurred during an interval of about 6 hours and stopped 2 to 3 hours before metaphase. After metaphase the chromosomes usually begin duplication again within 5 to 6 hours. Grain counting, to estimate the amount of tritium incorporated after a short contact with thymidine-H³ and at intervals after transfer to a medium with excess unlabeled thymidine, indicated that the intracellular pool of labeled precursors was diluted within less than a minute so that further labeling would not be detected. The chromosomes labeled during the contact period retained their precise pattern of labeling through another duplication cycle and no turnover of DNA or loss of tritium was detectable. Five or 6 chromosomes of the complement have segments typically late in duplication. Two of these are the X and Y chromosomes. The long arm of the X chromosome and the whole Y chromosome are duplicated in the last half of the interval of DNA synthesis. The short arm of the X chromosome in a male strain is duplicated in the first half of the interval. In another strain (female), one X chromosome had the same timing, but the other one was all duplicated in the last half of the period of DNA synthesis. The DNA in the short arms of 2 medium sized chromosomes, as well as most of the DNA in 1 or 2 of the smallest chromosomes of the complement was replicated late. The study has led to the hypothesis that various chromosomes or parts of chromosomes have a genetically controlled sequence in duplication which may have some functional significance.     

Types and frequencies of chromosomal aberrations induced by irradiation of different parts of interphase in Crepis capillaris

Quantitative and qualitative estimates of chromosomal damage in roots of Crepis capillaris were made in metaphase cells at many time intervals after irradiation with 200 or 400 rad of ⁶⁰Co gamma-rays. The results have confirmed the general pattern described for cells of other organisms, and have revealed in addition the following new facts. (1) The formation of aberrations of chromosome and chromatid type is not determined by the time of chromosome duplication alone. (2) The relative frequencies of different types of discontinuity form peaks with the following time succession: single gaps, chromatid breaks, isolocus breaks. (3) The location of peaks does not depend on the radiation dose, and shows no correlation which the time of synthesis. (4) Irradiation of G₂ induces a significant number of chromosome-type exchanges in Crepis. (5) Higher doses of radiation in G₂ favour the formation of chromatid over chromosome exchanges and of isochromatid breaks over chromosome breaks. A new interpretation of the production of certain types of aberration is discussed.     

Duplication of the bacteriophage lambda cohesive end site: genetic studies

A derivative of bacteriophage λ has been isolated that contains a duplication of the cohesive end site. To support this conclusion, the duplicated region has bean recovered by segregation from a lysogen of the duplication strain, and a derivative of the duplication strain was constructed that is heterozygous for the λ genes R and A, which bracket the cohesive end site. Duplication strains show no instability during lysogenization, suggesting that the virus particles each contain a single DNA molecule. During lytic growth, however, the strain is unstable and the duplication is frequently lost, even in the absence of all known recombination systems. Loss of the duplication is ascribed to cleavage of both cohesive end sites by the chromosome maturation system. Thus both cohesive end sites are functional, i.e. capable of being cleaved. No transfer of the duplicated region occurs in the absence of the known recombination systems. Thus, during λ chromosome maturation, cleavage of DNA molecules occurs but rejoining of cleaved molecules does not.     

Targeted Tandem Duplication of a Large Chromosomal Segment in Aspergillus oryzae

We describe here the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem chromosomal duplication was achieved by using strains that had a 5′-deleted pyrG upstream of the region targeted for tandem chromosomal duplication and a 3′-deleted pyrG downstream of the target region. Consequently, strains bearing a 210-kb targeted tandem chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the chromosomal duplication did not depend on the introduction of double-stranded breaks (DSBs) by I-SceI. The chromosomal duplications of these strains were stably maintained after five generations of culture under nonselective conditions. The strains bearing the tandem chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method.     

Natural Genetic Transformation Generates a Population of Merodiploids in Streptococcus pneumoniae

Partial duplication of genetic material is prevalent in eukaryotes and provides potential for evolution of new traits. Prokaryotes, which are generally haploid in nature, can evolve new genes by partial chromosome duplication, known as merodiploidy. Little is known about merodiploid formation during genetic exchange processes, although merodiploids have been serendipitously observed in early studies of bacterial transformation. Natural bacterial transformation involves internalization of exogenous donor DNA and its subsequent integration into the recipient genome by homology. It contributes to the remarkable plasticity of the human pathogen Streptococcus pneumoniae through intra and interspecies genetic exchange. We report that lethal cassette transformation produced merodiploids possessing both intact and cassette-inactivated copies of the essential target gene, bordered by repeats (R) corresponding to incomplete copies of IS861. We show that merodiploidy is transiently stimulated by transformation, and only requires uptake of a ∼3-kb DNA fragment partly repeated in the chromosome. We propose and validate a model for merodiploid formation, providing evidence that tandem-duplication (TD) formation involves unequal crossing-over resulting from alternative pairing and interchromatid integration of R. This unequal crossing-over produces a chromosome dimer, resolution of which generates a chromosome with the TD and an abortive chromosome lacking the duplicated region. We document occurrence of TDs ranging from ∼100 to ∼900 kb in size at various chromosomal locations, including by self-transformation (transformation with recipient chromosomal DNA). We show that self-transformation produces a population containing many different merodiploid cells. Merodiploidy provides opportunities for evolution of new genetic traits via alteration of duplicated genes, unrestricted by functional selective pressure. Transient stimulation of a varied population of merodiploids by transformation, which can be triggered by stresses such as antibiotic treatment in S. pneumoniae, reinforces the plasticity potential of this bacterium and transformable species generally.     

Specific chromosomal sites enhancing homologous recombination in Escherichia coli mutants defective in RNase H

To clone new replication origin(s) activated under RNase H-defective (rnh ^?) conditions in Escherichia coli cells, whole chromosomal DNA digested with EcoRI was to with a Km^r DNA fragment and transformed into an rnh^? derivative host. From the Kmr transformants, we obtained eight kinds of plasmid-like DNA, each of which contained a specific DNA fragment, termed “Hot”, derived from the E. coli genome. Seven of the Hot DNAs (HotA-G) mapped to various sites within a narrow DNA replication termination region (about 280 kb), without any particular selection. Because Hot DNA could not be transformed into a mutant strain in which the corresponding Hot region had been deleted from the chromosome, the Hot DNA, though obtained as covalently closed circular (ccc) DNA, must have arisen by excision from the host chromosome into which it had initially integrated, rather than by autonomous replication of the transformed species. While Hot DNA does not have a weak replication origin it does have a strong recombinational hotspot active in the absence of RNase H. This notion is supported by the finding that Chi activity was present on all Hot DNAs tested and no Hot-positive clone without Chi activity was obtained, with the exception of a DNA clone carrying the dif site.     

A 1.35 Mb DNA fragment is inserted into the DHMN1 locus on chromosome 7q34–q36.2

Distal hereditary motor neuropathies predominantly affect the motor neurons of the peripheral nervous system leading to chronic disability. Using whole genome sequencing (WGS) we have identified a novel structural variation (SV) within the distal hereditary motor neuropathy locus on chromosome 7q34–q36.2 (DHMN1). The SV involves the insertion of a 1.35 Mb DNA fragment into the DHMN1 disease locus. The source of the inserted sequence is 2.3 Mb distal to the disease locus at chromosome 7q36.3. The insertion involves the duplication of five genes (LOC389602, RNF32, LMBR1, NOM1, MNX1) and partial duplication of UBE3C. The genomic structure of genes within the DHMN1 locus are not disrupted by the insertion and no disease causing point mutations within the locus were identified. This suggests the novel SV is the most likely DNA mutation disrupting the DHMN1 locus. Due to the size and position of the DNA insertion, the gene(s) directly affected by the genomic re-arrangement remains elusive. Our finding represents a new genetic cause for hereditary motor neuropathies and highlights the growing importance of interrogating the non-coding genome for SV mutations in families which have been excluded for genome wide coding mutations.     

Buffering of Segmental and Chromosomal Aneuploidies in Drosophila melanogaster

Chromosomal instability, which involves the deletion and duplication of chromosomes or chromosome parts, is a common feature of cancers, and deficiency screens are commonly used to detect genes involved in various biological pathways. However, despite their importance, the effects of deficiencies, duplications, and chromosome losses on the regulation of whole chromosomes and large chromosome domains are largely unknown. Therefore, to explore these effects, we examined expression patterns of genes in several Drosophila deficiency hemizygotes and a duplication hemizygote using microarrays. The results indicate that genes expressed in deficiency hemizygotes are significantly buffered, and that the buffering effect is general rather than being mainly mediated by feedback regulation of individual genes. In addition, differentially expressed genes in haploid condition appear to be generally more strongly buffered than ubiquitously expressed genes in haploid condition, but, among genes present in triploid condition, ubiquitously expressed genes are generally more strongly buffered than differentially expressed genes. Furthermore, we show that the 4^th chromosome is compensated in response to dose differences. Our results suggest general mechanisms have evolved that stimulate or repress gene expression of aneuploid regions as appropriate, and on the 4^th chromosome of Drosophila this compensation is mediated by Painting of Fourth (POF).     

Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-Infected Bacillus subtilis I. Bacteriophage Deoxyribonucleic Acid Synthesis and Fate of Host Deoxyribonucleic Acid in Normal and Polymerase-Deficient Strains

The effect of bacteriophage SPO1 infection of Bacillus subtilis and a deoxyribonucleic acid (DNA) polymerase-deficient (pol⁻) mutant of this microorganism on the synthesis of DNA has been examined. Soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. This inhibition of host DNA synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate nucleotidohydrolase. Examination of the host chromosome for genetic linkage throughout the lytic cycle indicated that no extensive degradation occurred. After the inhibition of host DNA synthesis, a new polymerase activity arose which directed the synthesis of phage DNA. This new activity required deoxyribonucleoside triphosphates as substrates, Mg²⁺ ions, and a sulfhydryl reducing agent, and it was stimulated in the presence of adenosine triphosphate. The phage DNA polymerase, like that of its host, was associated with a fast-sedimenting cell membrane complex. The pol⁻ mutation had no effect on the synthesis of phage DNA or production of mature phage particles.