Chromosomal mapping of tRNA genes from Dictyostelium discoideum

Summary Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA^Val(GUU) and a tRNA^Val(GUA) gene. The method used involved a combination of classitics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA^Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F,), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VIII (A, H). Six tRNA^Val(GUA) genes from the NC4 isolate were mapped; one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).     

Stable-isotope analysis reveals sources of organic matter and ontogenic feeding shifts of a mangrove-dependent predator species,New Granada sea catfish,Ariopsis canteri

To gain a better understanding on the trophic ecology of New Granada sea catfish, Ariopsis canteri, and their linkage to mangroves, nitrogen and stable carbon isotopes (δ¹⁵N and δ¹³C), as well as Bayesian mixing models, were used to explore trophic dynamics and potential ontogenic feeding shifts across different size classes: class I (8–20 cm), class II (21–32 cm) and class III (>32 cm). The study area was the estuary of the Atrato River Delta, where information about fish ecology is scarce. The δ¹³C of size class I was lower (mean ± s.d . = −24.96 ± 0.69‰) than that of size classes II (−22.20 ± 0.90‰) and III (−22.00 ± 1.96‰). The δ¹⁵N of size class I was lower (mean ± s.d . = 8.50 ± 0.67‰) than that of size classes II (9.77 ± 0.60‰) and III (10.00 ± 0.66‰). Body size was positively and significantly correlated to δ¹⁵N and δ¹³C. Individuals with L_T > 32 cm presented the highest estimated trophic position (3.8). Five-source mixing models indicated that for class I, the mean estimated contribution of macroalgae was the highest (6%–57% c.i. ), and for classes II and III, the mean estimated contribution of macrophytes was the highest (3%–53% c.i. and 4%–53% c.i. , respectively). Ontogenetic feeding shifts of A. canteri were confirmed evidencing decreasing intraspecific competition between small and large individuals. Results suggest that mangroves are a nursery and feeding ground habitat for this species and that mangroves support A. canteri mainly due to the substrate/habitat that supports sources in the food webs. These results can be used in ecosystem-based fishery management focused on the protection of extensive mangrove areas in the southern Caribbean Sea.     

Recombination at the Rp1 locus of maize.

Summary The Rp1 locus of maize determines resistance to races of the maize rust fungus (Puccinia sorghi). Restriction fragment length polymorphism markers that closely flank Rp1 were mapped and used to study the genetic fine structure and role of recombination in the instability of this locus. Susceptible progeny, lacking the resistance of either parent, were obtained from test cross progeny of several Rp1 heterozygotes. These susceptible progeny usually had non-parental genotypes at flanking marker loci, thereby verifying their recombinational origin. Seven of eight Rp1 alleles (or genes) studied were clustered within about 0.2 map units of each other. Rpl ^G, however, mapped from 1–3 map units distal to other Rp1 alleles. Rp5 also mapped distally to most Rp1 alleles. Other aspects of recombination at Rp1 suggested that some alleles carry duplicated sequences, that mispairing can occur, and that unequal crossing-over may be a common phenomenon in this region; susceptible progeny from an Rp1 ^A homozygote had recombinant flanking marker genotypes, and susceptible progeny from an Rp1 ^DlRp1 ^F heterozygote showed both possible nonparental flanking marker genotypes.     

Physical mapping of genes on yeast mitochondrial DNA: Localization of antibiotic resistance loci, and rRNA and tRNA genes

Summary We have physically mapped the loci conferring resistance to antibiotics that inhibit mitochondrial protein synthesis (erythromycin, chloramphenicol and paromomycin) or respiration (oligomycin I and II), as well as the 21s and 14s rRNA and tRNA genes on the restriction map of the mitochondrial genome of the yeast Saccharomyces cerevisiae. The mitochondrial genes were localized by hybridization of labeled RNA probes to restriction fragments of grande (strain MH41-7B) mitochondrial DNA (mtDNA)¹ generated by endonucleases EcoRI, HpaI, BamHI, HindIII, SalI, PstI and HhaI. We have derived the HhaI restriction fragment map of MH41-7B mit DNA, to be added to our previously reported maps for the six other endonucleases.The antibiotic resistance loci (ant ^R) were mapped by hybridization of ³H-cRNA transcribed from single marker petite mtDNA's of low kinetic complexity to grande restriction fragments. We have chosen the single Sal I site as the origin of the circular physical map and have positioned the antibiotic loci as follows: C (99.5-1.Ou)-P(27-36.Ou)-O_II (58.3-62u)-O_I (80-84u)-E (94.4-98.4u). The 21s rRNA is localized at 94.4-99.2u, and the 14s rRNA is positioned between 36.2-39.8u. The two rRNA species are separated by 36% of the genome. Total mitochondrial tRNA labeled with ¹²⁵I hybridized primarily to two regions of the genome, at 99.5-11.5u and 34-44u. A third region of hybridization was occasionally detected at 70-76u, which probably corresponds to seryl and glutamyl tRNA genes, previously located to this region by petite deletion mapping.Supported by USPHS Training Grant T32-GM-07197.Supported by USPHS Training Grant 5-T01-GM-0090-19.The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U. S. Energy Research and Development Administration under Contract EY-76-C-02-0069.     

Genes involved in the biosynthesis of PQQ fromAcinetobacter calcoaceticus

From a gene bank of theAcinetobacter calcoaceticus genome a plasmid was isolated that complements four different classes of PQQ^- mutants. Subclones of this plasmid revealed that the four corresponding PQQ genes are located on a fragment of 5 kilobases. The nucleotide sequence of this 5 kb fragment was determined and by means of Tn5 insertion mutants the reading frames of the PQQ genes could be identified. Three of the PQQ genes code for proteins of M_r 29700 (gene I), M_r 10800 (gene II) and M_r 43600 (gene III) respectively. In the DNA region where gene IV was mapped however the largest possible reading frame encodes for a polypeptide of only 24 amino acids. A possible role for this small polypeptide will be discussed. Finally we show that expression of the four PQQ genes inAcinetobacter lwoffi andEscherichia coli lead to the synthesis of the coenzyme in these organisms.     

Linkage analysis of developmental mutations in aggregation-deficient mutants of Dictyostelium discoideum

Summary Simple parasexual genetic techniques have been employed to extend the linkage analysis initiated in an earlier study (Coukell, 1975) of developmental mutations (agg mutations) in 40 independently isolated aggregation-deficient mutants of Dictyostelium discoideum. Using these techniques, agg mutations in 28 of the 40 mutants have been assigned to 4 linkage groups: 16 in group II, 1 in group III, 10 in group IV, and 1 in group VI. None of the agg mutations analyzed appear to map in linkage group I. In addition, a new temperature-sensitive growth locus, designated tsgJ, was mapped in group III. It was also found that diploid strains of D. discoideum are readily induced to undergo haploidization when grown on 0.1% p-fluorophenylalanine (PFP) at 25.5 °C. Growth of diploid strains on PFP had no effect on the type of segregant classes obtained (i.e., PFP does not induce mitotic crossing-over), the subsequent growth and/or development of the segregants, or the ability of the segregants to reform stable diploids.     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

Impaired nitrogen fixation and glutamine synthesis in methionine sulfoximine sensitive (MSs) mutants of Rhizobium phaseoli

Summary Random Tn5 mutagenesis of antibiotic-resistant derivatives of Rhizobium phaseoli CFN42 yielded several independent mutants that were sensitive to methionine sulfoximine (MS^s), a specific inhibitor of glutamine synthetase (GS). These MS^s mutants were analyzed for GSI and GSII activities and for their symbiotic properties. Four classes of MS^s mutants have been distinguished. Class I strains are impaired in their synthesis of glutamine and in their symbiotic properties. Class II strains have wild type levels of GSI and GSII activities but have a reduced capacity to fix nitrogen. Class III strains have lost GSII activity, but their symbiotic properties are wild type. In class IV mutants neither glutamine synthesis nor symbiotic properties are affected. Mutants of classes I, III, and IV all have the Tn5 inserted into the chromosome, whereas in class II mutants the Tn5 is located in plasmid p42e, a plasmid different from the previously identified symbiotic plasmid p42d.     

RFLP-based genetic map of the homoeologous group 3 chromosomes of wheat and rye

Summary Genetic maps of chromosomes 3A, 3B and 3D of wheat and 3R of rye were developed using 22 DNA probes and two isozyme marker systems. Analysis of the 49 loci mapped showed extreme clustering around the centromere in all four maps, with large gaps in the distal chromosome regions, which is interpreted as being due to strong localisation of recombination towards the ends of the wheat and rye chromosomes. In the centromeric regions gene orders are highly conserved between the three wheat genomes and the rye genome. However, the unpredictable behaviour of the DNA clones that map in distal chromosome locations may indicate that the genomes are diverging most rapidly in the regions of higher recombination. A comparison of cDNA and genomic probes showed the latter to be much more efficient for revealing RFLP. Some classes of gDNA clones, i.e. chromosome-specific sequences and those hybridizing in a non-homoeologous manner, were seen to be most polymorphic. Correlations between map locations and RFLP levels showed no clear relationship. In addition to anonymous DNA clones, the locations of known function clones, sedoheptulose-1,7-bisphosphatase (XSbp), carboxypeptidase I (XCxp1) and a bZIP protein (XEmbp), were ascertained along with those for two isozyme loci, Mal-1 and Est-5.     

Mutagenesis by random cloning of an Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: Selection of mutants defective in photosynthesis

Summary Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation. This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Km^r) gene to restriction fragments of genomic DNA from the host. Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells. Several mutants impaired in photosynthesis were obtained by this procedure. All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I. Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Km^r gene into an AvaII restriction site of the gene psbD-1. All other harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant. A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping.     

Analysis of rice mitochondrial genome organization using pulsed-field gel electrophoresis

Most of the plant mitochondrial (mt) genomes that have been mapped are believed to be organized as master circle molecules from which sub-genomic molecules arise through homologous recombination. We have evidence to suggest that a major part of the rice mt genome is organized as independent, sub-genomic molecules or mt chromosomes, one of which has already been mapped. This study is aimed at the identification of the other molecular entities that comprise the genome. Pulsed-field gel electrophoresis of the native rice mt DNA and Southern analysis with different mt gene probes have shown that in addition to the 117 kb mt chromosome, at least four more such molecules of sizes 130 kb, 95 kb, 70 kb and 56 kb account for most of the rice mt genome. A majority of the rice mt genes that encode products involved in oxidative phosphorylation are distributed among these five chromosomes. Partial restriction map of the 95 kborf 25/cox 3 chromosome, indicating the sites for the enzymesBglII andHindIII has also been determined.     

In rhizobiaceae five different species are produced by rearrangements of one genome, induced by DNA-damaging agents

Summary All bacterial strains classified into the family Rhizobiaceae can be induced to undergo a fundamental genome rearrangement. The special structure of their genome allows the formation of five distinctive phenotypes, each one adapted to a different habitat (Fig. 1).This genome rearrangement can be induced by DNA-damaging agents, UV irridiation or chemical mutagenesis. For expression, cells have to be protected against photorepair and their replication has to be reduced by stress treatment. The rearrangement process is, with special exceptions, reversible. Classes I and II comprise Agrobacteria and Rhizobia, class III nitrogen-fixing strains and classes IV and V two different carotenoid-pigmented types. One of the class V strains has been shown to be an effective legume-symbiont. DNA characteristics and inter-class hybridization results show not only that the genomes are completely reconstructed during each step of rearrangement, but also that the bacteria of all five classes are genetically correlated. In many cases the genetic label has been maintained during rearrangement into the different classes. The identity of each class is protected by a class-specific restriction and modification system, which was analyzed by phage typing experiments and by functional analysis of class-specific restriction endonucleases. We propose to designate the classes as different species of Rhizobiaceae. The unidirectional rearrangement between nodulating Rhizobia and tumorgenic Agrobacteria has been interpreted as a sequence of decreasing complexity of genomic regions coding for the plant interactions of these bacteria.     

Mapping of additional restriction enzyme cleavage sites on bacteriophage MB78 genome

A physical map of bacteriophage MB78 DNA indicating the cleavage sites for the enzymeBglII,ClaI,EcoRI,PvuII,SalI andSmaI comprising of a total of 34 cleavage sites have been constructed earlier. The cleavage sites for a few more restriction endonucleases likeApaI,AvaI,BglI,HindIII,KpnI andXhoI have now been mapped. A total of 72 cleavage sites on MB78 DNA are known by now. Relative positions ofEcoRI I and J fragments which could not be decided earlier has now been determined.     

A physical map of the permuted genome of bacteriophage T1

Summary A restriction map has been constructed for the DNA of coliphage T1 which locates the cleavage sites of the restriction endonucleases, BglI (6 cuts), BglII (16 cuts), EcoRI (2 cuts), HindIII (2 cuts) and PstI (2 cuts). Digestions with BglI and BglII reveal fragments which are present in sub-molar quantities. Two methods, one using the selective removal of molecular ends with exonuclease III and the other involving the comparison of digestion patterns of concatemeric and virion DNA, have shown that the submolar fragments are at or close to the ends of the molecules. Digestions with BglI show that one terminal fragment has a very precise molecular weight whereas all the others are of heterogenous molecular weight. These results are consistent with the model for DNA packaging in which maturation is initiated at a precise site on a concatemeric precursor and proceeds by the encapsidation of up to four successive headfuls of 1.065 genome equivalents (MacHattie and Gill 1977).     

Primary productivity of the North Branch of the Raritan River,New Jersey

Summary Primary production of the North Branch of the Raritan River was measured at three sites by the diurnal oxygen curve method. An effluent of treated domestic sewage entered the river between the uppermost station (I) and the intermediate station (II). Mean gross productivity at Station I from May to September 1962 was 4.7 g O₂ m^–2 day^–1 (n = 8). In contrast, mean productivity from October 1961 1961 to September 1962 at Station II was 9.9 (n = 15), and at Station III it was 11.6 (n = 11). Efficiency of gross production based on estimated energy actually available to the community was from 1–7%.The daily maximum pH of river water was highly correlated with gross production, 24-hr community respiration and net daytime production at Stations II and III. Subject to further testing and refinement this may prove to be a simple but effective estimate of productivity in similar habitats.Chlorophyll a extract of periphyton growing on glass slides and small cement blocks were found to be statistically the same. Enumeration of these algal associations correlated significantly with the chlorophyll a extract at Stations II and III. However, these estimates of standing crop were not statistically correlated with primary production as determined from diurnal oxygen curves.Diatoms were the dominant organisms in most samples. During late summer the net plankton contained many planktonic blue-green algae. The periphytonwas characterized briefly during the spring by Ulothrix.Contribution No. 409 Natural Resources Institute University of Maryland.     

Solution structure and DNA binding of the catalytic domain of the large serine resolvase TnpX

The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site‐specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chloramphenicol resistance in the pathogens Clostridium perfringens and Clostridium difficile. TnpX consists of three structural domains: domain I contains the catalytic site, whereas domains II and III contain DNA‐binding motifs. We have solved the solution structure of residues 1–120 of the catalytic domain I of TnpX. The TnpX catalytic domain shares the same overall fold as other serine recombinases; however, differences are evident in the identity of the proposed hydrogen donor and in the size, amino acid composition, conformation, and dynamics of the TnpX active site loops. To obtain the interaction surface of TnpX_1–120, we titrated a DNA oligonucleotide containing the circular intermediate joint attCI recombination site into ¹⁵N‐labeled TnpX_1–120 and observed progressive nuclear magnetic resonance chemical shift perturbations using ¹⁵N HSQC spectra. Perturbations were largely confined to a region surrounding the catalytic serine and encompassed residues of the active site loops. Utilizing the perturbation map and the data‐driven docking program, HADDOCK, we have generated a model of the DNA interaction complex for the TnpX catalytic domain. Copyright © 2015 John Wiley & Sons, Ltd.     

Entwicklungsgenetische Untersuchungen an Gynandern vonDrosophila melanogaster

Summary Gynandromorphs with female XX-and male XO-areas result from the loss of an unstable ring-X-chromosome in the early cleavage mitoses of ring/rod-X-chromosome heterozygotes. The phenotypes of the recessive alleles on the rod-X-chromosome are expressed in the XO-areas.377 larval gynandromorphs of the genotypeR(1)2, In(1)w ^vC /y w sn³Iz^50e mal were examined and scored for the phenotypes of 13 paired and 10 unpaired structures (Table 2, Fig. 2). This was possible mainly by the cell-autonomous expression of aldehyde oxidase activity in soft tissues and by the comparison of the distribution of enzyme activity in wildtype and gynander larvae. The distances between pairs of structures were calculated in sturt-units (Tables 3 and 4). A morphogenetic fate map with the presumptive areas of larval structures was constructed (Fig. 3). The relative positions of the structures agree well with Poulson's fate map (Fig. 4). In addition, the distribution of phenotypes was scored in 380 adult gynandromorphs Table (5). The fate map (Fig. 5) which was constructed from these data is very similar to the fate map of larval structures. This similarity becomes even more pronounced if fate maps are constructed which contain only structures analogous in larva and imago (Table 6, Fig. 6). Therefore an attempt was made to set up an integrated morphogenetic fate map containing the presumptive areas of both larval and imaginal structures (Fig. 7). The possibilities of further blastoderm mapping are discussed.

Herrn Prof. Dr. Dr. h. c. B. Rensch zum 75. Geburtstag gewidmet     

Isolation and Partial Characterization of Four Host-specific Toxins of Helminthosporium maydis (Race T)

Helminthosporium maydis, race T, produces four host-specific toxins in culture. These have been designated toxins I, II, III, and IV. A method for isolation and purification of the four toxins is presented, and the criteria of purity of preparations of toxins I, II, and III are given. Toxins I and II are chemically similar and yield the same molecular ion when subjected to mass spectrometry, while toxin III appears to be a glycoside of a compound related to toxins I and II. Toxins I, II, and III can be biologically derived from ¹⁴C-mevalonic acid or ¹⁴C-acetate, permitting preparation of ¹⁴C-labeled toxins. Some chemical, spectral, and chromatographic properties of toxins I, II, and III are presented, and these data are discussed relative to the possible structure of the three compounds. In addition, four host-specific toxins have been isolated from corn infected with H. maydis (race T). These toxins are recovered in the same fractions as toxins I, II, III, and IV using the isolation procedure described here. Three of the toxins isolated from infected corn cannot be distinguished from toxins I, II, and III on the basis of infrared spectra or chromatographic mobility.     

Frequency of x-ray induced mitotic recombination in genotypes with different X-Y-combination-chromosomes in Drosophila melanogaster

Summary In first and second instar larvae of Drosophila melanogaster mitotic recombination has been induced by X-rays. Resulting mosaic spots were analysed in the eyes of adult females. The five examined genotypes varied in the combination of different X-chromosomes and in the presence or absence of the duplication Dp(1;3)N ^264-58. X-chromosomes used have been elongated by proximally or distally linked arms of the Y-chromosome. The portion of Y-heterochromatin in the genome is negatively correlated with the frequency of twin mosaic spots (Fig. 1). The frequency of these spots is lower in flies bearing the duplication than in those without the duplication. With respect to the X-chromosome combinations, there are no marked differences in the frequencies of single mosaic spots (Fig. 2); with respect to the duplication they are absent. In the genotype Y ^S X/X.Y ^S prophases of neuroblast mitoses (Fig. 4) show normal pairing of homologous chromosome sections. Reasons for the different spot frequencies are discussed.

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Vorgelegt von E. Hadorn     

Genetic Structure and Internal Rearrangements of Stable Merodiploids from BACILLUS SUBTILIS Strains Carrying the trpE26 Mutation

Transformation and transduction to tryptophan independence of strains of Bacillus subtilis carrying the "trpE26" chromosomal aberrations (a translocation and an inversion) with a "normal" 168 type strain as donor induce a tandem duplication of the thrA-ilvA region of the chromosome. The clones possessing this unstable duplication segregate besides the Trp^- some stable Trp⁺ cells which retain only part of the duplication (the trpE-ilvA region) in nontandem configuration. Such clones may also be produced directly during the crosses. The genetic map of these clones (designated as class I stable merodiploids) was constructed: they possess the tranlocation and the inversion of the trpE26 parental strain. Another type of stable Trp⁺ clones (class II) also appears, although more rarely, in similar crosses. Studies on their genetic structure revealed that they are haploid for the trpE-ilvA region and carry a nontandem duplication of the thrA-trpE region. In these clones the cysB-tre region has the orientation of the 168 type strain. The duplications in both classes are stable, that of class I being more stable than that of class II where loss of one copy of the thrA-trpE region leads to about 1% haploid cells. Detailed genetic studies on heterozygous clones from both classes have shown exchange of alleles between copies of the nontandem duplications. Models are proposed for the formation of each class of merodiploids and for recombination events taking place in them. These models imply recombination at sequences of intrachromosomal homology and (or) introduction of heterologous juncions ("novel joints") by transformation or transduction.