Telomeres and P-element of Drosophila melanogaster contain sequences that replicate autonomously in Saccharomyces cerevisiae

Summary We have isolated restriction fragments from a shotgun collection of Drosophila DNA which function as autonomously replicating sequences (ARS) in Saccharomyces cerevisiae and hybridize with telomeric regions of the 2L, 2R, 4, and X chromosomes. In an independently obtained set of D. melanogaster clones five fragments hybridize in situ with telomeres and a number of internal sites. Two of them also contain ARSs. A Drosophila mobile P-element also possesses ARS activity in yeast.     

RNA-linked nascent DNA pieces in T7 phage-infected Escherichia coli cells. I. Role of gene 6 exonuclease in removal of the linked RNA.

Summary The presence of RNA-linked nascent DNA pieces in T7 phage-infectedEscherichia coli cells has been shown by the selective degradation of the 5-hydroxyl-terminated nascent DNA, produced by alkali or RNase treatment, with spleen exonuclease. At 43°C, the proportion of RNA-linked DNA pieces in nascent short DNA is 50 to 60% in T7ts136 (ts mutant of gene 6) phage-infectedE. coli, whereas that in T7 wild-type phage-infected cells is less than 6%. Joining of the nascent pieces is greatly retarded in T7ts136-infectedE. coli temperature sensitivepolA mutants at 43° C. These results suggest that gene 6 exonuclease plays a role in removal of the linked RNA during the discontinuous replication of T7 DNA.     

Size distribution and molecular polarity of nascent DNA in a temperature-sensitive dna G mutant of Escherichia coli

Summary In the dna G t.s. strain BT 308, made lysogenic for the phage , nascent DNA was labelled by short pulses of ³H-thymidine, isolated and separated as a function of size by alkaline sucrose gradient sedimentation. The molecular polarity of the labelled DNA was then determined by hybridization to the separated strands of DNA.At 30° C, strand r DNA, made in the direction opposite that of fork movement, is synthesized in the form of short pieces. The first observable consequences of a shift to 42° C are the preferential inhibition of strand r synthesis and the small amount of strand r DNA which is made is recovered in long pieces of DNA rather than in short fragments. This indicates that the t.s. product, in strain BT 308, may be involved in the synthesis of the strand growing in the direction opposite that of replication fork movement.Newly synthesized strand l DNA, made in the same direction as replication fork movement, is found in long pieces in wild-type bacteria; it is found in pieces of intermediate size in strain BT 308 at 30° C as well as at 42° C. This indicates additional differences in the replication machinery between strain BT 308 and wild-type bacteria.     

Host genes involved in the replication of single-stranded DNA phage ?K

Summary Using various replication mutants of E. coli, the host genes that participate in the replication of some K12-specific single-stranded DNA phages have been determined. Functional products of dnaE,-F,-G and -Z genes are required for the multiplication of K, whereas dnaA,-B,-C(D), H,-I and -P are dispensable for viral replication. In contrast with polB, recA, B, C, or xth functions, host rep activity is essential for K. At the restrictive temperature, the yield of K was markedly reduced in the ligts7 mutant and partially decreased in a polA ^ts strain. The phage K is thus less dependent on the host cells than X174 and A which require additionally the dnaB,-C(D) and -H functions. Replication of phage St-1 depends on dnaG and -Z gene products, but not on dnaP function. Although not much affected in polA ^ts host, growth of St-1 was significantly diminished in dnaF or ligts7 mutants.     

Fluorescence polarization of stretched polytene chromosomes stained with acridine orange

The molecules of the fluorescent dye acridine orange (AO) bind to DNA in such a way that the absorption and emission dipoles lie on a plane perpendicular to the DNA axis. For this reason, definite fluorescence polarization should correspond to each mode of spatial DNA packing. A chromosome, considered as an axially symmetrical ensemble of DNA, was characterized by two experimental parameters, P and P , i.e., by polarizations of fluorescence excited by light polarized parallel and perpendicular to the symmetry axis. In view of the sequential order in the packing levels of DNA fiber in a chromosome, it was suggested that, under mechanical stretching, the highest level is disrupted first, then the others, in the order of their sequence.Isolated chromosomes of Chironomus thummi were stained with AO and stretched with needles of a micromanipulator. From the changes of P and P measured during stretching it was concluded the polytene chromosome bands have three, at least, DNA packing levels, tentatively described as 100 å fiber, 250 å coil and chromomere.     

Multivariate morphometric study of theCardamine pratensis group (Cruciferae) in the Carpathian and Pannonian area

A multivariate morphometric study of theCardamine pratensis group is presented, based on 84 population samples collected from the Carpathian and Pannonian area in the Czech Republic, Slovakia, Poland, Ukraine, Hungary, and Romania. Among the multivariate methods, principal component analysis, cluster analysis, and classificatory and canonical discriminant analysis were used. The analysis of chromosome numbers from all populations studied showed wide variation. The morphometric study showed that not all groups of populations characterised by their chromosome numbers and geographical criteria are morphologically, and thus taxonomically, distinguishable. Besides the morphologically well characterised speciesCardamine dentata andC. rivularis, the following species were recognised in the area studied:C. matthioli, C. majovskii andC. pratensis. Within the last species, besides the typical populations, two diploid types are provisionally recognised: type ucranica and type rivularis auct..     

Characterization of two temperature-sensitive mutants of Sigma virus (authors transl)

Summary Temperature-sensitive mutants have been isolated from Drosophila Sigma virus, a Rhabdovirus inducing CO₂ sensitivity in Drosophila melanogaster. We have studied the decay of infectious centers at non permissive temperature. The proportion of destroyed infectious centers is the same for the wild type, ts⁺, and for ts9. On the opposite, it is more important for ts 4. Temperature-sensitive function of ts 4 appears necessary to the viral genome replication. With the three clones, ts⁺, ts 4 and ts 9, we have obtained stabilized Drosophila females able to transmit Sigma virus to their whole progeny. We have tried to see in each case, if stabilized flies could transmit the virus to their progeny at non permissive temperature. Flies stabilized with ts⁺ and ts 9 can, flies stabilized with ts 4 cannot. Therefore two categories of mutants are defined: those that are transmitted hereditarily. at non permissive temperature, and not blocked in genome replication. Those that are blocked in genome replication and not transmitted. When the virus cannot replicate, the divisions in the germ line cells dilute the viral genomes. The consequence will be a real healing of germ line cells, and then a break in hereditary transmission by stabilized flies. All the results with temperature-sensitive mutants are coherent with this hypothesis.

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Étude de mutants thermosensibles du virus Sigma

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Mémoire présenté par F. Gros     

DNA sequences homologous to the T DNA region of Agrobacterium tumefaciens are present in diverse Rhizobium species

Summary DNA sequences homologous to the T DNA region of the octopine-type Ti plasmid from Agrobacterium tumefaciens are present in different Rhizobium species. Plasmid DNA from each of two R. leguminosarum, two R. meliloti, and four slow-growing Rhizobium strains examined contain restriction endonuclease fragments that hybridize with the T DNA region, or with DNA sequences at or near the adjacent Ti plasmid transfer (ra) region. Four different BamHI fragments that contain homology to the T DNA region were cloned from R. leguminosarum 300 plasmid DNA. Cloned fragments of 5.9 kb and 10.3 kb hybridize to each other and are homologous to sequences which map at the right boundary region (EcoRI fragment 24) of the core T DNA. Ti plasmid sequences homologous to those present in cloned fragments of 10.9 kb and 2.0 kb map in adjacent fragments near the tra genes, approximately 10 kb to the right of the core T DNA.     

The role of DNA polymerase I, II and III in the replication of the bacteriocinogenic factor Clo DF13

Summary The replication of the bacteriocinogenic factor Clo DF13 was studied in Escherichia coli mutants which lack either DNA polymerase I (polA1 and resA1 mutants), DNA polymerase II (polB1 mutant) or DNA polymerase III (dnaE mutant). DNA polymerase I is required for Clo DF13 replication. The Clo DF13 factor, however, can be maintained in a strain carrying the polA107 mutation and thus lacking the 53 exonucleolytic activity of DNA polymerase I. DNA polymerase II is not required for transfer replication and maintenance of the Clo DF13 plasmid. In the temperature sensitive dnaE mutant, Clo DF13 can replicate at the nonpermissive temperature during the first two hours after the temperature shift from 30°C to 43°C. During this period DNA polymerase III seems not to be essential for Clo DF13 replication.     

Replicon size of yeast ribosomal DNA

Summary The ribosomal RNAs of the yeast Saccharomyces cerevisiae are transcribed from a 9Kbp stretch of DNA which is reiterated about 120-fold in a continuous array, about 360 m long, on chromosome XII. Although ARS activity has been detected in the repeat unit, the size and disposition of replicons along this array of identical genes has not hitherto been determined. We have used immobilised rRNA as a probe to examine the size of radioactively labelled rDNA replicons resolved on alkaline sucrose gradients. The replicons were found to be uniformly sized, about 5 repeat units in length, and groups of 4 adjacent replicons may be activated simultaneously. These observations suggest that replicon initiation events are not determined solely by the recognition of specific DNA sequences that function as origins of replication.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Summary In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the simultaneous mechanism the cotransporterC forms a ternary complexNCS with Na⁺ and the substrateS; coupled transport of Na⁺ andS involves a conformational transition between statesNCS andNCS with inward- and outward-facing binding sites. In the consecutive (or ping-pong) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the simultaneous model, but incompatible with the consecutive mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the simultaneous model can be estimated from the experimental results. The equilibrium dissociation constants of Na⁺ and alanine at the extracellular side are determined to beK _N <-64mm andK _S <-18mm. Furthermore, the ratioK _N /K _N ^S of the dissociation constants of Na⁺ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na⁺ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na⁺ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.     

Diploid state of phenotypically recombinant progeny arising after protoplast fusion in Bacillus subtilis

Summary After fusion of Bacillus subtilis protoplasts the phenotypically recombinant clones isolated, whether immediately or as segregants of complementing diploid clones, have in common the following properties. They appear independently of the recN ⁺ gene, most often as the result of apparently non-reciprocal recombination occurring in genetic intervals encompassing the origin and the terminus of replication. First indicated by reciprocal fusion crosses between 105-lysogenic and 105-sensitive strains, the diploidy of the recombinants was confirmed by studying the transforming activities of their DNA. These experiments establish heterozygosity at eight loci scattered on the chromosome map. By revealing the presence of the rpF ⁺ allele in trpF7 recombinants, the results also strongly suggest that stable phenotypic recombinants may arise by genetic inactivation. Two possible genetic structures for these recombinants are discussed, one implying total inactivation of one recombinant chromosome, the other a segmentary inactivation of one unrecombined chromosome. Whatever the structure, genetic stability is not a reliable sign of haploidy in bacterial clones produced after protoplast fusion.     

Replication forks are not found in a Drosophila minichromosome demonstrating a gradient of polytenization

Differential DNA replication is widely held to influence polytene chromosome structure by causing the dramatic reductions in heterochromatic DNA content that are characteristic of most endopolyploid cells. The underreplication model of heterochromatic sequence underrepresentation predicts that replication intermediates should populate regions of DNA between fully polytenized euchromatic sequences and underpolytenized heterochromatic sequences. We directly tested this prediction using Dp1187, a 1300 kb Drosophila minichromosome containing well-defined heterochromatic regions. DNA from a euchromatic/heterochromatic junction region of Dp1187, demonstrating a significant gradient of underrepresentation in larval salivary glands, lacked the stalled replication forks predicted by the underreplication model. We consider an alternative mechanism leading to heterochromatic sequence underrepresentation involving a process of DNA elimination.by W. Hennig     

Hemocyte responses to implanted tissues inDrosophila melanogaster larvae

Summary At 26° C temperature-sensitivetu(1) Sz ^ts larvae ofDrosophila melanogaster develop melanotic tumors consisting of aberrant caudal adipose tissue encapsulated by precociously differentiated hemocytes (lamellocytes). Whentu-Sz ^ts larvae are grown at 18° C, lamellocytes are present but the caudal fat body surfaces remain normal and melanotic tumors do not develop (Rizki and Rizki, preceding paper). In this paper we demonstrate that the lamellocytes intu-Sz ^ts larvae at 18° C encapsulate implants of mechanically-damaged fat bodies and adipose cells devoid of basement membrane, while leaving host fat bodies or implanted fat bodies with intact basement membrane unencapsulated. Therefore, low temperature blocks melanotic tumor formation by normalizing the surfaces of the prospective tumor-forming sites intu-Sz ^ts.The discriminatory ability oftu-Sz ^ts lamellocytes was examined by challenging them with undamaged heterospecific tissues. Tissues from sibling species ofD. melanogaster were not encapsulated whereas tissues fromDrosophila species outside theD. melanogaster species subgroup were. Ultrastructural examination of encapsulated heterospecific tissues showed intact basement membrane, so we propose that distinction between self and not self by lamellocytes depends upon the molecular architecture of the basement membrane. In similar series of experiments usingD. virilis donor tissues inOre-R wild type larval hosts, fat bodies remained unencapsulated and imaginal disks metamorphosed. These studies suggest that continued presence of lamellocytes in the larval host is a prerequisite for encapsulation.     

The frequency of the γ chain variant AγT in different populations,and its use in evaluating γ gene expression in association with thalassemia

Summary The occurrence of the ^AT chain (i.e. ^A75 Ile Thr) in different populations was evaluated through a study of 4250 cord blood samples and blood samples from more than 350 SS¹ patients. High frequencies were observed in Italy, Yugoslavia, Turkey, Holland, but also in Japan, Vietnam, and India. The chain is (nearly) absent in the Black population of Ghana and Kenya, and low frequencies were observed in China and Australian aborigines. Only a few adult SS patients (18 out of 357) were ^AT heterozygotes. The chromosomes with the ^AT globin gene were mapped through an evaluation of the presence of 10 different restriction sites. The ^AT chromosomes from different populations were closely related and had the same subhaplotypes of [--++-+] (Hinc II 5 to ; Xmn I 5 to ^G; Hind III in ^G and ^A; Hinc II in and 3 to ), quite different from the subhaplotypes seen for ^AT negative chromosomes.² This suggests a common ancestor which may have originated in Southern Europe. An evaluation of the chain production by both chromosomes in SS patients and -thalassemia heterozygotes was possible for subjects with an ^AT heterozygosity. It was concluded that in -thalassemia trait, the chain synthesis is directed for about two-thirds by the thalassemic chromosome and for about onethird by the normal chromosome; the contribution by the normal chromosome decreases with a decrease in total chain production.This is contribution #0890 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA     

Control of DNA replication and spatial distribution of defined DNA sequences in salivary gland cells of Drosophila melanogaster

In dividing cells, each sequence replicates exactly once in each S-phase, but in cells with polytene chromosomes, some sequences may replicate more than once or fail to replicate during S-phase. Because of this differential replication, the control of replication in polytene cells must have some unusual features. Dennhöfer (1982a) has recently concluded that the total DNA content of the polytene cells of Drosophila salivary glands exactly doubles in each S-phase. This observation, along with previous studies demonstrating satellite underreplication in salivary gland cells, led us to consider the hypothesis that there is a doubling of DNA mechanism for the control of DNA replication in polytene cells. With this mechanism, a doubling of DNA content, rather than the replication of each sequence, would signal the end of a cycle of DNA replication. To test this hypothesis, we have reinvestigated the replication of several sequences (satellite, ribosomal, histone and telomere) in salivary gland cells using quantitative in situ hybridization. We find that underreplication of some sequences does occur. In addition we have repeated Dennhöfer's cytophotometric and labeling studies. In contrast to Dennhöfer, we find that the total DNA contents of nonreplicating nuclei do reflect this partial replication, in accord with Rudkin's (1969) result. We conclude that DNA replication in polytene cells is controlled by modifications of the mechanism operating in dividing cells, where control is sequence autonomous, and not by a doubling of DNA mechanism. — In situ hybridization to unbroken salivary gland nuclei reveals the distribution of specific sequences. As expected, satellite, histone and 5S sequences are usually in a single cluster. This rules out the possibility that sequences known to be underreplicated in chromosomal DNA exist as extrachromosomal copies. Telomere sequences are grouped into two to six clusters, as if the chromosome ends are partially but not completely paired in salivary gland nuclei.     

Chromosomal locations of the gene coding for the CD3 (T3) γ subunit of the human and mouse CD3/T-cell antigen receptor complexes

The gene coding for the M _r 26000 chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 -2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse cDNA clone pB10.AT3 -1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 genes have been described previously. Thus, the corporate results indicate that the CD3 and genes have remained together since they duplicated about 200 million years ago.     

Acinetobacter calcoaceticus liberates chromosomal DNA during induction of competence by cell lysis

A transformation assay was used to assay the amount of DNA present in the extracellular medium of a growing culture of Acinetobacter calcoaceticus. It was observed that small amounts of DNA were liberated during the entire exponential growth phase in a batch culture. Release of DNA could be fully accounted for by lysis of cells. Lysis was quantified via simultaneous measurement of -galactosidase activity of cells and supernatant, with a strain that contained a plasmid (pAPA100) with lacZ under control of a constitutive -lactamase promoter. In conclusion, no evidence could be obtained indicating that Acinetobacter calcoaceticus actively excretes DNA, to be used for DNA exchange.     

Characterization of the dnaA, gyrB and other genes in the dnaA region of the Escherichia coli chromosome on specialized transducing phages lambda tna.

Summary Specialized transducing phages tna (tryptophanase) harboring chromosomal DNA and genetic markers from the dnaA region of the Escherichia coli chromosome were isolated. Transductional analysis showed that some of these tnaA transducing phages carry two genes important in DNA replication, namely the dnaA gene (initiation of chromosome replication) and the gyrB gene (subunit B of DNA gyrase), formerly designated cou ^R. The following clockwise order of genetic markers was found: uhp, gyrB, dnaA, rimA, tnaA, bglB.The gene-protein relationship was established by the determination of the gene products encoded on the chromosomal DNA of the different tna. A 54 kD and a 91 kD polypeptide appear to be coded for by the dnaA and gyrB genes, respectively; the 91 kD protein is encoded on a region in which coumermycin sensitivity maps and is with respect to electrophoretic behavior identical to subunit B of DNA gyrase. The 54 kD protein is encoded on the region in which different independently isolated dnaA(Ts) mutations (dnaA5, dnaA46, dnaA167, dnaA203, dnaA204, dnaA205, dnaA211, dnaA508) are located. Additional genes which code for polypeptides with hitherto unknown functions were identified and mapped. The acriflavin sensitivity mutation acrB1 was found to be an allele of the gyrB gene (see Note Added in Proof).     

Comparative effects of CGA-92194, cyometrinil,and flurazole on selected metabolic processes of isolated soybean leaf cells

The potential adverse phytotoxic effects of the herbicide safeners CGA-92194 {-[1,3-dioxolan-2-yl-methoxy)imino]benzeneacetonitrile}, cyometrinil [-(cyanomethoxy)imino-benzeneacetonitrile] and flurazole [phenylmethyl 2-chloro-4-(trifluoromethyl)-5-thiazole-carboxylate] on selected metabolic processes of enzymatically isolated leaf cells of soybean [Glycine max (L.) Merr.] were compared in time- and concentration-course studies. CO₂ fixation, protein synthesis, RNA synthesis, DNA synthesis, and lipid synthesis were assayed by the incorporation of NaH¹⁴CO₃, [¹⁴C]-leucine, [¹⁴C]-uracil, [³H]thymidine, and [¹⁴C]-acetate, respectively, into the isolated cells. CGA-92194 and cyometrinil behaved similarly, and at low concentrations (0.1, 1, and 10 M) they stimulated rather than inhibited the five metabolic processes assayed, following incubation periods of up to 2 h. At the highest concentration of 100 M, both safeners inhibited all metabolic processes of the soybean leaf cells but neither compound exhibited rapid and distinct inhibitions as might be expected in the case of inhibition of a primary target site by a potent inhibitor. At low concentrations and early incubation periods (30 and 60 min), flurazole effects on all metabolic processes were also stimulatory rather than inhibitory. However, the stimulation of CO₂ fixation by 0.1 and 1.0 M was highly significant. At 100 M flurazole was extremely potent on all metabolic processes of soybean leaf cells examined. At the 2-h incubation period, flurazole also inhibited all metabolic processes at concentrations lower than 100 M. The sensitivity of the five metabolic processes to flurazole decreased in the following order: photosynthesis = lipid synthesis >DNA synthesis>protein synthesis>RNA synthesis.Contribution No. 534, Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg VA 24061 USA.