A DNA sequence of Drosophila melanogaster with a differential telomeric distribution

A DNA sequence (8–19T) of 2.3 kilobase pairs (kb) of Drosophila melanogaster was localized by in situ hybridization to the extreme ends of polytene chromosomes and to the chromocenter. The relative abundance of this sequence at the ends of polytene chromosomes X2L2R3L3R is 13.41.902.7. This differential distribution is probably due to different copy numbers at the individual telomeric regions. Restriction enzyme analysis of genomic DNA shows that 8–19T sequences are interspersed with other sequences. The clone 8–19T, which contains most of this interspersed repetitive sequence, is itself not internally repetitive but has a complex sequence composition. Some of these sequences are transcribed into poly(A)⁺RNA. We suggest that the ends of Drosophila chromosomes are of a complex arrangement with some sequences common to all ends.     

Genetic relationships among actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin

Summary A polyphasic taxonomic study was undertaken to establish the genetic and phenotypic relationships among six actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin. Chemotaxonomic studies reveal that all have Type I cell walls. Gas chromatography (GC) of fatty acid methyl esters revealed patterns consistent for strains ofStreptomyces with 160 and 150anteiso predominating. Principal component analysis of GC data revealed distinct profiles for each culture. Reciprocal DNA homology studies atT _m -25 showed the rapamycin-producing strain and one FK506-producing strain to have 38–50% homology with the type strain ofStreptomyces hygroscopicus (ATCC 27438). The remaining strains exhibited 6–17% homology. To further explore the relationships among these strains all were probed for the presence of anO-methyltransferase gene specific to this biosynthetic pathway. Among the strains of interest, onlyStreptomyces hygroscopicus subsp.yakushimaensis, the patent strain for FK520/FK523, failed to hybridize with the probes.     

Yeast PAPS reductase: properties and requirements of the purified enzyme

The enzymatic mechanism of sulphite formation in Saccharomyces cerevisiae was investigated using a purified 3-phosphoadenylsulphate (PAPS) reductase and thioredoxin. The functionally active protein (M_R 80–85 k) is represented by a dimer which reduces 3-phosphoadenylyl sulphate to adenosine-3,5-bisphosphate and free sulphite at a stoichiometry of 1:1. Reduced thioredoxin is required as cosubstrate. Examination of the reaction products showed that free anionic sulphite is formed with no evidence for bound-sulphite(s) as intermediate. V _max of the enriched enzyme was 4–7 nmol sulphite · min^-1 · mg^-1 using the homologous thioredoxin from yeast. The velocity of reaction decreased to 0.4 nmol sulphite · min^-1 · mg^-1 when heterologous thioredoxin (from Escherichia coli) was used instead. The K _m of homologous thioredoxin was 0.6 · 10^-6 M, for the heterologous cosubstrate it increased to 1.4 · 10^-6 M. The affinity for PAPS remained practically unaffected (K _{m PAPS}: 19 · 10^-6 M in the homologous, and 21 · 10^-6 M in the heterologous system). From the kinetic data it is concluded that the enzyme followed an ordered mechanism with thioredoxin as first substrate followed by PAPS as the second. Parallel lines in the reciprocal and a common intersect in the Hanes-plots for thioredoxin were seen as indication of a ping-pong (with respect to thioredoxin) uni-bi (with respect to PAPS) mechanism.Abbreviations APS adenylyl sulphate - DTE dithioerythritol - DTT dithiothreitol - HPLC high performance liquid chromatography - IEF isoelectric focusing - LSC liquid scintillation counting - 3,5-PAP adenosine-3,5-bisphosphate - PAPS 3-phosphoadenylyl sulphate - PEP phospho-(enol)pyruvate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - Tris 2-amino-2-hydroxymethyl-1,3-propanediol     

DNA sequence analysis of spontaneous mutation in a PolA1 strain of Escherichia coli indicates sequence-specific effects

Summary The sequences of a collection of 261 spontaneous lacI^- mutants recovered in a PolA^- strain of Escherichia coli have indicated an increase in the frequency of most classes of mutation in this strain. Among base substitutions in lacI, a preference for transversions over transitions was observed. In addition, a single transition in the lac operator was enhanced 8-fold. More significantly, of 18 frameshifts, 12 occurred adjacent to a 5-GTGG-3 sequence. Likewise, 15 of 24 deletions and 2 of 10 duplications had 5-GTGG-3 sequences at one or both endpoints. We speculate that the prevalence of mutations at these specific sequences reflects the persistence of strand discontinuities that enhance the opportunity for mutagenic mishaps. Further, 5-GTGG-3 sequences apparently represent sites where DNA polymerase I is involved in some aspect of DNA metabolism. These results strengthen the view that DNA context contributes an important component to spontaneous mutagenesis and indicate an anti-mutagenic role for DNA polymerase I.     

Decreased DNA content of birch (Betula) chromosomes at high ploidy as determined by cytophotometry

Relative amounts of DNA were determined on telophase nuclei by Feulgen cytophotometry for euploid taxa of birch (Betula) with somatic chromosome numbers of 28, 42, 56, 70, and 84. A direct correlation was found between observed DNA absorbance and chromosome number except for plants of B. papyrifera with 84 somatic chromosomes. The DNA density value for nuclei of the 84-chromosome plants fitted a 12.25 ratio instead of the expected 13.0 ratio. The DNA density value for these plants was calculated to be approximately equivalent to plants which would possess 63 somatic chromosomes. The average DNA value per chromosome was 2.73 for the 84-chromosome plants in contrast to 3.50 per chromosome in each of the lower euploids. Nuclear diameters of the 84-chromosome plants were directly related to chromosome number and not to DNA density value. The genomic number of Betula was considered to be x=7, rather than x=14, since a DNA value equivalent to 63 chromosomes is a multiple of 7 and not 14. Diploid birch species (2n=2x=28), therefore, would actually be tetraploids (2n=4x=28). The reduction in DNA content may be an adaptation for the establishment of higher ploidy in birches.     

Towards an integrated linkage map of common bean 2. Development of an RFLP-based linkage map

Summary A restriction fragment length polymorphism (RFLP)-based linkage map for common bean (Phaseolus vulgaris L.) covering 827 centiMorgans (cM) was developed based on a F₂ mapping population derived from a cross between BAT93 and Jalo EEP558. The parental genotypes were chosen because they exhibited differences in evolutionary origin, allozymes, phaseolin type, and for several agronomic traits. The segregation of 152 markers was analyzed, including 115 RFLP loci, 7 isozyme loci, 8 random amplified polymorphic DNA (RAPD) marker loci, and 19 loci corresponding to 15 clones of known genes, 1 virus resistance gene, 1 flower color gene, and 1 seed color pattern gene. Using MAPMAKER and LINKAGE-1, we were able to assign 143 markers to 15 linkage groups, whereas 9 markers remained unassigned. The average interval between markers was 6.5 cM; only one interval was larger than 30 cM. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios (121 or 31) and mapped into four clusters. Probes of known genes belonged to three categories: seed proteins, pathogen response genes, and Rhizobium response genes. Within each category, sequences homologous to the various probes were unlinked. The I gene for bean common mosaic virus resistance is the first disease resistance gene to be located on the common bean genetic linkage map.     

Distribution and composition of particulate organic matter in the Ross Sea (Antarctica)

The biochemical composition and spatial distribution of particulate organic matter (POM) were studied in the Ross Sea (Antarctica) in summer 1989 to assess the quantitative role of organic carbon fractions in the cycling of organic matter in the water column. Large differences in chemical composition were observed between surface and deep layers. The results indicated that, despite large geographical differences, POM was quite homogeneous, of phytoplankton origin and mostly detrital. Different ratios were used to investigate the changes in biochemical composition of particulate organic matter in relation to the ice-melting: CN (organic carbonorganic nitrogen ratio) and C-POMPOC (sum of carbohydrate, protein and lipid carbontotal organic carbon ratio) were used to analyse the percentage of refractory organic material. PPRTPCHO (proteincarbohydrate ratio) were used to establish POM age and RNADNA ratios as a relative measure of particulate activity; POCChl a and N-PPRTChl a ratios were used to estimate the autotrophic contribution to the suspended particulate organic matter. Despite its low caloric value (5.3 Kcal g POM^–1), an high caloric content in the photic layer (1.6 Kcal m^–3 of POM and 2.5 Kcal m^–3 of POC) was found thus indicating that a large amount of food was available to higher trophic levels.     

Contrasting response of euchromatin and heterochromatin to translocation in polytene chromosomes of Drosophila melanogaster

Studies on Feulgen-DNA content in the polytene chromosomes of D. melanogaster T(14)w ^m258-21 heterozygotes showed that when the euchromatic region 3D₁-E₂ is located next to the heterochromatic breakpoint it contains less DNA than in the non-translocated homologue (Hartmann-Goldstein and Cowell, 1976). In contrast to the region adjacent to the breakpoint, region 3C_1–10, which contains intercalary heterochromatin, shows more DNA in the translocated than in the non-translocated chromosome. Transposition may induce morphologically euchromatic regions containing putatively underreplicated sequences to undergo additional replication cycles. Region 2E₁-3A₄, distal to 3C₁ and at some distance from the heterochromatic breakpoint is apparently unaffected. Extended replication and reduced DNA content in regions which have undergone chromosomal rearrangement could be accounted for by varying degrees of blockage of replication in individual strands of the polytene chromosome.     

Meiotic segregation of five different reciprocal translocations in the onion fly,Hylemya antiqua (Meigen)

For one translocation (T14) with short interstitial segments in Hylemya antiqua significant differences in segregation behaviour between males and females were observed. In males the ratio of alternate:adjacent 1:adjacent 2 was approximately 730 and in females about 813. This difference is attributed to the difference in type of chromosome association. Female meiosis is chiasmate and male meiosis is achiasmate. It is suggested that meiotic pairing in males results in relative short Coorientation Determing Distances (CDDs) between homologous centromeres which favours alternate and adjacent 1 segregation. In females because of non-localized chiasmata on the average no differences in CDD between homologous and nonhomologous centromeres are expected. This might explain the occurrence of coorientation between non-homologous centromeres resulting in adjacent 2 segregations. Four other translocations with longer interstitial segments than T14 showed in males as well as females predominantly an alternate and adjacent 1 segregation, adjacent 2 was hardly found (0–3.6%). The longer distance between non-homologous centromeres is probably the reason.     

Cytogenetic investigations in rye,wheat and triticale

Summary Chromosomes of tetra- and hexaploid wheat have been individually characterized by Giemsa and/or Leishman C-banding techniques. Appropriate methodological modifications resulted in almost identical staining of chromosomes of tetraploid wheat with Giemsa and Leishman solutions. Additionally comparison of Giemsa banded chromosomes of the A- and B-genome of Triticum turgidum 34 and Triticum aestivum cv Jubilar reveals similar or corresponding patterns in all homologous chromosomes with the exception of chromosome 7B. Apart from this intervarietal variation in certain homologous chromosomes of both wheat cultivars, intravarietal polymorphism is verified.     

Autoradiographic studies of the human Y chromosome

An autoradiographic analysis (using continuous labeling with tritiated thymidine) was made on 317 cells from four normal males. The labeling pattern of the Y chromosome was compared to the first and the last chromosomes to complete replication as well as to G21–22. The Y chromosome was never found to be the last chromosome in the cell to complete replication. Instead, it completed DNA synthesis relatively early (usually among the first 10 chromosomes) but had a distinctively heavy label during the earliest stages of late-S. In 51% of those cells with one labeled G+Y chromosome, a G21–22 was labeled and the Y was not.—It was concluded, therefore, that the human Y chromosome is not a late-replicating chromosome but terminates replication earlier than most of the autosomes. In addition, the Y chromosome cannot be distinguished from the G chromosomes on the basis of a consistent and differential labeling pattern.Supported by USPHS Grant GM 15361.     

DNA polymorphisms in the ψβ1- and β-globin gene regions in asian macaques

DNA polymorphisms in the ₁--globin gene region in nine Asian macaques(Macaca fuscata, M. mulatta, M. nemestrina, M. cyclopis, M. fascicularis, M. arctoides, M. radiata, M. maura, andM. assamensis) were examined using several restriction endonucleases and the human ₁, IVS2, and IVS2 probes. TheBamHI site 3 to the -globin gene was polymorphic inM. fuscata andM. mulatta, while the HincII site and the EcoRI site in the ₁-globin gene region was highly polymorphic inM. fuscata andM. mulatta, respectively. These polymorphic sites also seem to be present in other Asian macaques. The present study of the polymorphism at theBamHI site 3 to the -globin gene in Asian macaques supports, at the nuclear DNA level, the idea that thefascicularis group includingM. fuscata, M. mulatta, M. cyclopis, andM. fascicularis is different from other Asian macaque groups.This study was supported in part by the Cooperation Research Program of the Primate Research Institute, Kyoto University.     

A preliminary genetic analysis of TE146, a very large transposing element of Drosophila melanogaster

TE146 is a transposing element (TE) consisting of six polytene chromosome bands that has inserted into the no-ocelli (noc 250) locus. This member of Ising's TE family carries two copies of the white and roughest loci. TE146 is lost from noc with a spontaneous frequency of approximately 1 in 22000 chromosomes. All spontaneous losses are accompanied by the reversion of the noc mutation associated with the TE. The TE is associated with fold-back (FB) sequences. The losses of TE146 retain fold-back homology at noc. Of 26 -ray-induced losses of TE146, 16 are gross deletions, removing loci neighboring noc and ten are not. The non-deleted -ray-induced losses are either noc ^– and rst ⁺ or noc ⁺ and rst ^–. The white⁺ genes of TE146 are dosage compensated since w/Y; TE146/+ and w/w; TE146/+ flies are sexually dimorphic for eye color. These w ⁺ genes are also suppressed by zeste since z w; TE146/+ flies have zeste-colored eyes.     

Polymorphism and stability in the histone gene cluster of Drosophila melanogaster

Histone genes in Drosophila melanogaster are organized into repeats of 4.8 and 5.0 kb (Lifton et al., 1978). We find these repeat sizes in every one of the more than 20 Drosophila strains we have examined. Strains differ in the relative amounts of the two repeat types, with ratios varying from 11 to 14, the 5.0 kb repeat always present in equal or greater amounts than the 4.8 kb repeat. Restriction enzyme digestion and blotting analysis reveals that the strains also differ in a number of far less abundant fragments containing histone DNA sequences. In the Amherst and Samarkand strains, there are, in addition, many copies of 4.0 and 5.5 kb repeat-like fragments respectively. A series of stocks were made isogenic for single second chromosomes from the Amherst strain. The hybridization patterns of the histone DNA from these stocks containing different Amherst chromosomes are very similar but a number of differences in the minor fragments were seen. The stability of the histone locus restriction pattern was tested by following the DNA derived from a single second chromosome of the b Adhn² pr cn strain over a two year period. The restriction pattern of major and minor bands remained identical. Finally, histone loci distinguishable by their restriction pattern on blots were recombined with visible markers. These chromosomes will be useful in tracing the fate of specific histone loci during genetic manipulations.     

The yeast,Saccharomyces cerevisiae,RNase P/MRP ribonucleoprotein endoribonuclease family

Ribonuclease P (RNase P) is a ribonucleoprotein responsible for the endonucleolytic cleavage of the 5-termini of tRNAs. Ribonuclease MRP (RNase MRP) is a ribonucleoprotein that has the ability to cleave both mitochondrial RNA primers presumed to be involved in mitochondrial DNA replication and rRNA precursors for the production of mature rRNAs. Several lines of evidence suggest that these two ribonucleoproteins are related to each other, both functionally and evolutionarily. Both of these enzymes have activity in the nucleus and mitochondria. Each cleave their RNA substrates in a divalent cation dependent manner to generate 5-phosphate and 3-OH termini. In addition, the RNA subunits of both complexes can be folded into a similar secondary structure. Each can be immunoprecipitated from mammalian cells with Th antibodies. In yeast, both have been found to share at least one common protein. This review will discuss some of the recent advances in our understanding of the structure, function and evolutionary relationship of these two enzymes in the yeast,Saccharomyces cerevisiae.Abbreviations LRI long range interaction - mt mitochondrial - MRP mitochondrial RNA processing - NME nuclear mitochondrial endonuclease - POP processing of precursor - RNase ribonuclease - SNM suppressor of NME - RNP ribonucleoprotein     

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     

Replication of DNA in larval salivary glands of Drosophila after in vivo synchronization

The replication of DNA in the giant chromosomes in different cells of Drosophila larval salivary glands is asynchronous. A method of in vivo synchronization of the nuclei has been successfully devised by a 5-fluorodeoxyuridine (FdU) block-release-thymidine chase technique, and the patterns of replication sequences have been examined by ³H-thymidine autoradiography. When the larvae of Drosophila melanogaster are fed on FdU for 48 h, and the block is released thereafter, most cells are found in mid-replication phase (termed 3C). When the larvae are subjected to a chase in normal Drosophila medium (or sucrose), a series of cells arrive at 3C phase about every 8 h. When they are chased in sucrose containing thymidine, the number of cells in 3C phase rises to 70%, and then drops rapidly to 1–2% of all labelled cells. The terminal phases (3D, 2D and 1D) reach a peak between 4–8 h. At 12–14 h of chase the 3D-1D peaks decline and a third peak consisting mostly of the initial phases (DD-1C) is found at 14–16 h. The replication of DNA in polytene chromosomes of Drosophila thus seems to proceed in a regular sequence of DD-3C-1D.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Establishment of molecular markers and linkage groups in two F2 populations of Upland cotton

Two F₂ populations of cotton (Gossypium hirsutum L.) from the crosses of HS46 x MARCABUCAG8US-1-88 (MAR) and HS46 x Pee Dee 5363 (PD5363) were characterized for restriction fragment length polymorphisms (RFLPs) using DNA probes. Seventy-three probe/enzyme combinations were used in the HS46 x MAR population analysis, which resulted in 42 informative polymorphic fragments. These 42 moleclar markers represented 26 polymorphic loci, which consisted of 15 codominant and 11 dominant (+/-) genotypes. Chi-square analyses of these loci fit expected genotypic ratios of 121 and 31, respectively An analysis of these loci with the MAPMAKER program resulted in the establishment of four linkage groups A, B, C, and D with 4,2,2, and 2 loci, respectively, as well as 16 unlinked loci. Six probe-enzyme combinations were assayed on the HS46 x PD5363 population, which resulted in 11 informative polymorphic fragments. These 11 fragments represented 6 polymorphic loci, 1 dominant (+/-) and 5 codominant genotypes. The MAPMAKER analysis of these loci yielded 2 linked loci. Thus, a total of 53 polymorphic fragments and 32 polymorphic loci, representing five linkage groups, were identified among the two families.Contribution of the USDA-ARS in cooperation with the Miss Agric For Exp Stn.     

Localization of the polymorphic human calcitonin gene on chromosome 11

Summary A molecular probe containing a 584 base pairs sequence corresponding to part of the human calcitonin mRNA was used for the chromosomal assignment of the calcitonin gene. Restriction endonuclease analysis of DNA from human-Chinese hamster and human-mouse somatic cell hybrids, including some containing a translocation of human chromosomes, placed the calcitonin gene in the p14qter region of chromosome 11.Analysis of human DNA showed that the calcitonin gene has a polymorphic site for restriction endonuclease TaqI.