DNS-Replikation und die Natur der spät replizierenden Orte im X-Chromosom von Drosophila melanogaster

Zusammenfassung Der Verlauf der DNS-Replikation im X-Chromosom von Drosophila melanogaster und im besonderen in der white-Region wurde mit Hilfe der ³H-Thymidin-Autoradiographie untersucht. Die Anzahl der markierten Kerne in den Speicheldrüsen schwankt zwischen 20 und 50%; dabei überwiegen die Partialmarkierungsmuster der Chromosomen. Im einzelnen wurden fünf verschiedene Markierungstypen unterschieden, zwei mit kontinuierlicher und drei mit Partialmarkierung. Die fünf Markierungstypen lassen sich zwanglos in eine Reihe einordnen, die mit der kontinuierlichen Markierung beginnt und durch die immer weiter abnehmende Anzahl der markierten spots definiert ist.Die Lokalisation der Markierung legt allgemein den Schluß nahe, daß die Querscheiben Replikationseinheiten sind. Bewiesen ist dies aber nicht. Es scheint auch kein einfacher Zusammenhang zwischen Querscheibendicke und Zeitdauer der DNS-Replikation zu bestehen. Die Frequenz und Intensität der Markierung, die beide als Parameter des Replikationsverhaltens bestimmt wurden, zeigen im allgemeinen eine positive Korrelation. Die lokale Markierungsintensität scheint gegen Ende der Replikationsphase allmählich abzusinken.Die Regionen mit der größten Markierungsfrequenz (lange oder spät replizierende Orte) zeigen die charakteristischen Eigenschaften des interkalaren Heterochromatins (z. B. ectopic pairing) und sind mit den bereits bekannten weak points identisch. Mit Hilfe verschiedener w-Defizienzen konnte die Lage des spätreplizierenden Materials in der Region 3C präzisiert werden. Es liegt cytologisch zwischen den Querscheiben 3C3 und 3C6 und genetisch zwischen den Loci von w und rst. Das spätreplizierende Material selbst scheint keine bekannten Gene zu enthalten.Bei w-Duplikationen mit veränderter zeste-white-Interaktion und ihren Derivaten wurden quantitative Änderungen im DNS-Replikationsverhalten des weak point in 3C festgestellt. Die möglichen Ursachen derartiger Veränderungen, Vermehrung oder Vergrößerung der DNS Replikationseinheiten, werden diskutiert.

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DNA replication and the nature of late replicating loci in the x-chromosome of Drosophila melanogaster

The course of DNA replication in the male salivary gland X chromosome of Drosophila melanogaster and the nature of the late replicating spots have been investigated by means of ³H-thymidine radioautography combined with cytological and cytogenetic studies. Following a 5 minutes pulse, between 20% and 50% of the salivary gland nuclei were found labeled, with the majority (80%) showing discontinuous labeling patterns. Five principal labeling patterns of the male X can be distinguished, 2 continuous and 3 discontinuous ones. These can be ordered into a sequence beginning with total labeling and ending with labeling restricted to a few spots usually situated in regions 3C, 11A, and 12DE. Both the frequency and the intensity of labeling were scored for all late replicating spots. The two parameters seem to be positively correlated with each other. Intensity gradually declines towards the end of the replication period of each spot.Late replication behavior can in all cases be associated cytologically with one or two, sometimes three or four, closely adjacent relatively heavy bands, but there is no direct relation between the apparent duration of replication and band thickness in different spots. However, most of the very late replicating spots show the characteristics of interstitial heterochromatin (ectopic pairing, weak spot behavior). Using various white deficiencies the property of late replication in region 3C could be associated with the interval 3C3 to 3C5, corresponding to the segment between white and roughest in the genetic map.The correlation, as observed in some duplications and rearrangements, between changes in the interaction between zeste and white and changes of replication behavior of the 3C spot is considered as accidental. Possible models to explain changes of labeling intensity and frequency are discussed.

     

Local repeat sequence organization of an intergenic spacer in the chloroplast genome ofChlamydomonas reinhardtii leads to DNA expansion and sequence scrambling: A complex mode of “copychoice replication”?

Parent-specific, randomly amplified polymorphic DNA (RAPD) markers were obtained from total genomic DNA ofChlamydomonas reinhardtii. Such parent-specific RAPD bands (genomic fingerprints) segregated uniparentally (through mt⁺) in a cross between a pair of polymorphic interfertile strains ofChlamydomonas (C. reinhardtii andC. minnesotti), suggesting that they originated from the chloroplast genome. Southern analysis mapped the RAPD-markers to the chloroplast genome. One of the RAPD-markers, “P2” (1.6 kb) was cloned, sequenced and was fine mapped to the 3 kb region encompassing 3′ end of 23S, full 5S and intergenic region between 5S and psbA. This region seems divergent enough between the two parents, such that a specific PCR designed for a parental specific chloroplast sequence within this region, amplified a marker in that parent only and not in the other, indicating the utility of RAPD-scan for locating the genomic regions of sequence divergence. Remarkably, the RAPD-product, “P2” seems to have originated from a PCR-amplification of a much smaller (about 600 bp), but highly repeat-rich (direct and inverted) domain of the 3 kb region in a manner that yielded no linear sequence alignment with its own template sequence. The amplification yielded the same uniquely “sequence-scrambled” product, whether the template used for PCR was total cellular DNA, chloroplast DNA or a plasmid clone DNA corresponding to that region. The PCR product, a "unique" new sequence, had lost the repetitive organization of the template genome where it had originated from and perhaps represented a “complex path” of copy-choice replication.     

Cloning and characterization of thewhite andtopaz eye color genes from the sheep blowflylucilia cuprina

Summary Clones carrying thewhite andtopaz eye color genes have been isolated from genomic DNA libraries of the blowflyLucilia cuprina using cloned DNA from the homologouswhite andscarlet genes. respectively, ofDrosophila melanogaster as probes. On the basis of hybridization studies using adjacent restriction fragments, homologous fragments were found to be colinear between the genes from the two species. The nucleotide sequence of a short region of thewhite gene ofL. cuprina has been determined, and the homology to the corresponding region ofD. melanogaster is 72%; at the derived amino acid level the homology is greater (84%) due to a marked difference in codon usage between the species. A major difference in genome organization between the two species is that whereas the DNA encompassing theD. melanogaster genes is free of repeated sequences. that encompassing theirL. cuprina counterparts contains substantial amounts of repeated sequences. This suggests that the genome ofL. cuprina is organized on the short period interspersion pattern. Repeated sequence DNA elements, which appear generally to be short (less than 1 kb) and which vary in repetitive frequency in the genome from greater than 10⁴ copies to less than 10² copies, are found in at least two different locations in the clones carrying these genes. One type of repeat structure, found by sequencing, consists of tandemly repeating short sequences. Restriction site and restriction fragment length polymorphisms involving both thewhite andtopaz gene regions are found within and between populations ofL. cuprina.     

Fluctuations during growth of the plasma membrane H(+)-ATPase activity of Saccharomyces cerevisiae and Schizosaccharomyces pombe

The plasma membrane H⁺-ATPase activity was determined under various growth conditions using the yeastsSaccharomyces cerevisiae andSchizosaccharomyces pombe. Under early batch-growth conditions in a rich medium, the budding yeastS. cerevisiae ATPase specific activity increased 2-to 3-fold during exponential growth. During late exponential growth, a peak of ATPase activity, followed by a sudden decrease, was observed and termed “growth-arrest control”. The growth arrest phenomenon ofS. cerevisiae could not be related to the acidification of the culture medium or to glucose exhaustion in the medium or to variation of glucose activation of the H⁺-ATPase. Addition of ammonium to a proline minimum medium also stimulated transiently the ATPase activity ofS. cerevisiae. Specific activity of the fission yeastS. pombe ATPase did not show a similar profile and steadily increased to reach a plateau in stationary growth. Under synchronous mitotic growth conditions, the ATPase activity ofS. cerevisiae increased during the cell division cycle according to the “peak” type cycle, while that ofS. pombe was of the “step” type.     

Patterns of protein synthesis in salivary glands ofDrosophila melanogaster during larval and prepupal development

Summary Patterns of protein synthesis in the salivary glands ofDrosophila melanogaster have been studied throughout late larval and prepupal development by pulse labelling the tissues with³⁵S-methionine. Specific changes to the pattern of proteins synthesized during development are found and the significance of these changes is discussed in view of the known changes in gene (puffing) activity which occur at the same times. We review the problem of salivary gland function in prepupalDrosophila.     

FB elements can promote exon shuffling: a promoter-less white allele can be reactivated by FB mediated transposition in Drosophila melanogaster

Foldback (FB) elements are transposable elements found in many eukaryotic genomes; they are thought to contribute significantly to genome plasticity. In Drosophila melanogaster, FBs have been shown to be involved in the transposition of large chromosomal regions and in the genetic instability of some alleles of the white gene. In this report we show that FB mediated transposition of w ^67C23, a mutation that deletes the promoter of the white gene and its first exon, containing the start codon, can restore expression of the white gene. We have characterized three independent events in which a 14-kb fragment from the w ^67C23 locus was transposed into an intron region in three different genes. In each case a local promoter drives the expression of white, producing a chimeric mRNA. These findings suggest that, on an evolutionary timescale, FB elements may contribute to the creation of new genes via exon shuffling.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. P. Georgiev     

Mobilization of a Hobo-related Sequence in the Genome of Drosophila simulans

The hobo transposable element can occur under three forms in the Drosophila genome: as a complete element (also called canonical), as internally deleted copies, or as hobo-related sequences (relics). Some evidence indicated that canonical elements and internally deleted copies are recent acquisitions of Drosophila genomes, while the “relics” are old components, normally degenerated and immobile. Here we present the characterization of a hobo-related sequence, found in the genome of a hypermutable strain of D. simulans, which insertion into the white locus raised a de novo white mutation. It is a shorter hobo related element presenting, overall, roughly 18% of divergence at the DNA level from the canonical hobo, with many indels that make clear this element is defective. However, its ITRs and flanking regions are extremely conserved. This is the first hobo “relic” showed to be mobilizable. We suggest, and point up some evidences, toward the idea that this sequence could have been mobilized by the canonical element. The presence of a similar “relic” element in D. sechellia allows us to suggest that these elements have been maintained mobilizable since the time of divergence between these species.     

Structural feature of sorghum chloroplast psbA gene and regulation effects of its 5'-noncoding region*

Comparative analysis reveals remarkahle homology between the sequences of bothpsbA gene nucleotides and the inferred amino acids of sorghum, a C₄ plant, and those of rice, a C₃ plant. The 5′-noncoding region of sorghumpsbA gene contains the conservative promoter elements, “—35” element and “—10” element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using anin vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically hinds to the 5′-noncoding region ofpsbA gene. Measurement of the expression of luciferase shows a 2–5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghumpsbA gene than that of the expression plasmids pALqr which contain leader region of ricepsbA gene inE. coli. Project supported by the Chinese National “863” and “973” Projects     

Genetic instability in Drosophila melanogaster. Evidence for insertion mutations

Summary An X chromosome in Drosophila melanogaster is described which is mutationally unstable. Mutational events were identified through phenotypic changes associated with a tandem duplication of the X chromosome in which the white locus is present in duplicate. The left segment of the tandem duplication was marked with the mutant w ^sp, the right segment with mutant w ^17G. Some of the phenotypic changes were identified as deletions involving the w ^17G marked segment of the duplication. Other phenotypic changes involved the left segment in which phenotypically w ^sp mutated to w. Experimental evidence is presented which attributes these latter mutations to insertions of foreign DNA into the w locus equivalent to the insertion mutations of E. coli.     

Characteristics of stress response in a mushroom-pathogenic bacterium,Pseudomonas tolaasii, during the interaction withPleurotus ostreatus and carbon/nitrogen starvation in vitro

A brown blotch bacterium,Pseudomonas tolaasii strain PT814, expresses a high degree of cross-protection against generalized stress imposed by physical/chemical treatment, H₂O₂, UV, high temperature, ethanol and NaCl during the interaction withPleurotus ostreatus. Stress resistance was also noted in the bacterium in vitro under limited carbon and nitrogen sources. In addition, changes in cell morphology from a “metabolically active” rod to an “energy-saving” spherical shape were detected during starvation and the interaction. All the changes under stress were reversible. A homologue ofrpoS (σ ^S), a regulator that controls such physiological status during starvation in other bacteria, was identified inP. tolaasii strain PT814. Data suggest that the bacterium is able to withstand a complex stress environment for its survival through changes in its metabolic pattern.     

Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25° C and β-heterochromatic in X0 males at 14° C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin. Received: 1 July 1998 / Accepted: 7 September 1998     

A new petunidin tetraglycoside and tulipanin fromOphiopogon seeds

Blue seed-coats ofOphiopogon jaburan have been found to contain two kinds of anthocyanins. By means of paper chromatographic and spectral analyses, one present as a minor component was determined to be delphinidin 3-rutinoside, tulipanin, and the major component, a new anthocyanin, was identified as petunidin 3-O-β-(2^G-glucosylrutinoside)-5′-glucoside, which the authors have named “ophionin”. Both anthocyanins were also present in the blue seed-coasts ofO. japonicus andO. planiscapus.     

Preliminary study of taxonomy ofAzotobacter andAzomonas by using rocket line immunoelectrophoresis

Rocket line immunoelectrophoresis was used to study the taxonomy ofAzotobacter andAzomonas assessed by reaction with antiserum to the AVO₂ strain ofAzotobacter. Forty-five cultures, comprising seventeen species in five genera, showed that antigen “β”, like high-titer somatic agglutination, was restricted to all 11 strains ofAzotobacter vinelandii and to one strain which has been namedAzotobacter macrocytogenes (10EM). A thermoresistant antigen (“γ”) was found to be shared by all strains and species ofAzotobacter andAzomonas investigated.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

Metabolism ofAedes aegypti cells grown in vitro. 1. Incorporation of3H-uridine and14C-leucine

Summary Metabolic activity ofA. aegypti cells grown in vitro has been studied by incorporation of³H-uridine and¹⁴C-leucine. “Chase” experiments with unlabeled precursors, and the use of actinomycin D and puromycin, showed that³H-uridine was incorporated into cellular RNA, and that¹⁴C-leucine was incorporated into protein of these cells. Incorporation of³H-uridine was inhibited when actinomycin D was used at a concentration of 10 μg/ml, and¹⁴C-leucine incorporation was inhibited to the same extent by puromycin at a concentration of 100 μg/ml medium. Contribution No. 148.     

Differential excision patterns of the <Emphasis Type="Italic">En</Emphasis>-transposable element at the <Emphasis Type="Italic">A2</Emphasis> locus in maize relate to the insertion site

Defined mutant alleles with resident transposons display characteristic patterns of germinal and somatic reversion, and heritable changes in the timing and frequency of reversions, which have been termed “change of state” by McClintock, constantly arise. Several mechanisms were proposed to account for these changes. They may be ascribed to the structure and composition of the elements themselves (composition hypothesis) or to their location (position hypothesis). In the current study, insertion positions were determined for three autonomous En-controlled mutable alleles of the A2 locus in maize that show different somatic reversion patterns. A relationship was observed between En insertion positions in the single coding region of the intronless A2 gene and anthocyanin variegation patterns in the aleurone. An insertion in the 5′ region of the coding sequence produced a very late somatic variegation pattern, whereas two early variegation patterns were caused by En insertions in the 3′ region of the coding sequence.     

Rapid shifting of foraging pattern by Yakushima macaques ( Macaca fuscata yakui ) in response to heavy fruiting of Myrica rubra

We describe short-term changes in foraging behavior by wild Yakushima macaques (Macaca fuscata yakui),which inhabit a warm-temperate broad—leaved forest on Yakushima Island (30°N, 131°E), Japan. Rapid changes of dietary composition, activity budget, and range use by the monkeys occurred from May to June, apparently associated with changes in the availability of the fruit of Myrica rubraBefore the fruit ripened, monkeys spent less time moving and more time feeding on many species of leaves, which accounted for 40% of feeding time. However, when M. rubrabegan to ripen, they fed intensively on the fruit, which accounted for three-fourths of feeding time,though the activity budget remained unaffected As fiuit of M. rubradecreased,the monkeys fed more on the fruit of other species and on insects, and spent more time moving at higher speeds. There marked shifts in foraging pattern occurred within only two months. In terms of moving cost and dietary quality,Yakushima macaques shifted their foraging pattern according to the availability of M. rubrafrom a “low-cost, low-yield” strategy to a “low-cost, high-yield” strategy, and then to a more costly strategy. The ability to make such rapid shifts in foraging pattern may allow the macaques to effectively use the highly variable food supply within their small range.     

Correspondence of banding patterns to 3H-thymidine labeling patterns in polytene chromosomes

³H-thymidine labeling frequencies over X chromosomal region 1A-4E of Drosophila melanogaster, were analysed with reference to chromosome sections with and without prominent bands. A correspondence was found between band sections and late start of silver grain labeling at the initial stage in combination with late labeling at the end stage of replication. A complementary situation is always to be found over puff/interband sections, where an early start of labeling at the initial stage is generally combined with early labeling completion at the end stage of replication.     

Conformational preferences of modified nucleoside N(2)-methylguanosine (m(2)G) and its derivative N(2), N(2)-dimethylguanosine (m(2)(2)G) occur at 26th position (hinge region) in tRNA

Conformational preferences of the modified nucleosides N²-methylguanosine (m²G) and N², N²-dimethylguanosine (m₂²G) have been studied theoretically by using quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. Automated complete geometry optimization using semiempirical quantum chemical RM1, along with ab initio molecular orbital Hartree–Fock (HF-SCF), and density functional theory (DFT) calculations has also been made to compare the salient features. Single-point energy calculation studies have been made on various models of m²G26:C/A/U44 and m₂²G26:C/A/U44. The glycosyl torsion angle prefers “syn” (χ = 286°) conformation for m²G and m₂²G molecules. These conformations are stabilized by N(3)–HC2′ and N(3)–HC3′ by replacing weak interaction between O5′–HC(8). The N²-methyl substituent of (m²G26) prefers “proximal” or s-trans conformation. It may also prefer “distal” or s-cis conformation that allows base pairing with A/U44 instead of C at the hinge region. Thus, N²-methyl group of m²G may have energetically two stable s-trans m²G:C/A/U or s-cis m²G:A/U rotamers. This could be because of free rotations around C–N bond. Similarly, N², N²-dimethyl substituent of (m₂²G) prefers “distal” conformation that may allow base pairing with A/U instead of C at 44th position. Such orientations of m²G and m₂²G could play an important role in base-stacking interactions at the hinge region of tRNA during protein biosynthesis process.     

Effect of abscisic acid on betacyanin leakage from plant tissues

Effect of abscisic acid on cell permeability in leaves ofIresine u allisi hort. and roots ofBeta vulgaris L. were examined. An increase of betacyanin leakage from leaf cells was shown by ABA at 10⁻⁴, 10⁻⁷ or 10⁻⁹ M concentrations in water solution at 25 °C. The efflux of batacyanin from tissues did not change during the joint action of ABA and PEG 1000. ABA could lower the betacyanin leakage fromIresine leaves and beet-root slices under severe osmotic stress, as was found by deplasmolysis. The results suggest that ABA elicits some alteration in density of tonoplast membranes under dehydration. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.