Su(UR)ES: A gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes

A genetic locus suppressing DNA underreplication in intercalary heterochromatin (IH) and pericentric heterochromatin (PH) of the polytene chromosomes of Drosophila melanogaster salivary glands, has been described. Found in the In(1)sc^V2 strain, the mutation, designated as Su(UR)ES, was located on chromosome 3L at position 34.8 and cytologically mapped to region 68A3-B4. A cytological phenotype was observed in the salivary gland chromosomes of larvae homozygous and hemizygous for Su(UR)ES: (i) in the IH regions, that normally are incompletely polytenized and so they often break to form “weak points,” underreplication is suppressed, breaks and ectopic contacts disappear; (ii) the degree of polytenization in PH grows higher. That is why the regions in chromosome arm basements, normally β-heterochromatic, acquire a distinct banding pattern, i.e., become euchromatic by morphological criteria; (iii) an additional bulk of polytenized material arises between the arms of chromosome 3 to form a fragment with a typical banding pattern. Chromosome 2 PH reveals additional α-heterochromatin. Su(UR)ES does not affect the viability, fertility, or morphological characters of the imago, and has semidominant expression in the heterozygote and distinct maternal effect. The results obtained provide evidence that the processes leading to DNA underreplication in IH and PH are affected by the same genetic mechanism.     

Nucleolar dominance in polytene cells of Drosophila

Previous studies indicate that genes from only one of the cell's nucleolus organizers undergo multiple rounds of DNA replication in polytene cells of Drosophila. This report presents evidence that this effect is mediated by a function that is associated with the ribosomal genes of the dominant or replicating X chromosome. This function can act in trans to result in replication of the ribosomal genes on the recessive X chromosome in flies that are bobbed for the dominant X chromosome. In these cases, ribosomal genes from both chromosomes undergo polytenization. Heterochromatic regions that flank the nucleolus organizer have little or no effect on nucleolar dominance. In addition, deletion of the compensatory response (cr⁺) locus does not affect the dominance, suggesting that ribosomal gene compensation and nucleolar dominance in polytene cells of Drosophila are separate genetic phenomena.     

High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants

The structural and functional analyses of heterochromatin are essential to understanding how heterochromatic genes are regulated and how centromeric chromatin is formed. Because the repetitive nature of heterochromatin hampers its genome analysis, new approaches need to be developed. Here, we describe how, in double mutants for Su(var)3-9 and SuUR genes encoding two structural proteins of heterochromatin, new banded heterochromatic segments appear in all polytene chromosomes due to the strong suppression of under-replication in pericentric regions. FISH on salivary gland polytene chromosomes from these double mutant larvae allows high resolution of heterochromatin mapping. In addition, immunostaining experiments with a set of antibodies against euchromatic and heterochromatic proteins reveal their unusual combinations in the newly appeared segments: binding patterns for HP1 and HP2 are coincident, but both are distinct from H3diMetK9 and H4triMetK20. In several regions, partial overlapping staining is observed for the proteins characteristic of active chromatin RNA Pol II, H3triMetK4, Z4, and JIL1, the boundary protein BEAF, and the heterochromatin-enriched proteins HP1, HP2, and SU(VAR)3-7. The exact cytological position of the centromere of chromosome 3 was visualized on salivary gland polytene chromosomes by using the centromeric dodeca satellite and the centromeric protein CID. This region is enriched in H3diMetK9 and H4triMetK20 but is devoid of other proteins analyzed.     

Drosophila P elements preferentially transpose to replication origins

The P transposable element recently invaded wild Drosophila melanogaster strains worldwide. A single introduced copy can multiply and spread throughout the fly genome in just a few generations, even though its cut-and-paste transposition mechanism does not inherently increase copy number. P element insertions preferentially target the promoters of a subset of genes, but why these sites are hotspots remains unknown. We show that P elements selectively target sites that in tissue-culture cells bind origin recognition complex proteins and function as replication origins. The association of origin recognition complex-binding sites with selected promoters and their absence near clustered differentiation genes may dictate P element site specificity. Inserting at unfired replication origins during S phase may allow P elements to be both repaired and reduplicated, thereby increasing element copy number. The advantage transposons gain by moving from replicated to unreplicated genomic regions may contribute to the association of heterochromatin with late-replicating genomic regions.     

The complete sequence of the 1,683-kb pSymB megaplasmid from the N2-fixing endosymbiont Sinorhizobium meliloti

Analysis of the 1,683,333-nt sequence of the pSymB megaplasmid from the symbiotic N(2)-fixing bacterium Sinorhizobium meliloti revealed that the replicon has a high gene density with a total of 1,570 protein-coding regions, with few insertion elements and regions duplicated elsewhere in the genome. The only copies of an essential arg-tRNA gene and the minCDE genes are located on pSymB. Almost 20% of the pSymB sequence carries genes encoding solute uptake systems, most of which were of the ATP-binding cassette family. Many previously unsuspected genes involved in polysaccharide biosynthesis were identified and these, together with the two known distinct exopolysaccharide synthesis gene clusters, show that 14% of the pSymB sequence is dedicated to polysaccharide synthesis. Other recognizable gene clusters include many involved in catabolic activities such as protocatechuate utilization and phosphonate degradation. The functions of these genes are consistent with the notion that pSymB plays a major role in the saprophytic competence of the bacteria in the soil environment.     

Clustered tRNA genes in Schizosaccharomyces pombe centromeric DNA sequence repeats.

The centromere-associated B' and B DNA sequence repeats of Schizosaccharomyces pombe chromosomes I and II have been found to contain clusters of tRNA genes. The centromere II region (cen2) includes at least 22 tRNA genes distributed among five copies of the B sequence repeat containing genes specifying tRNA(Ile), tRNA(Ala), and tRNA(Val). Individual B repeats are variously associated with other tRNA genes, including those specifying tRNA(Lys), tRNA(Arg), and tRNA(Glu2). The centromere I region (cen1) contains at least six tRNA genes in two copies of the B' repeated element, including genes specifying tRNA(Ile), tRNA(Ala), and tRNA(Glu3). Multiple tandemly arranged clusters of tRNA genes are presumably conserved due to restricted recombination frequencies in the centromere regions.     

Site-specific carcinogen binding to DNA in polytene chromosomes.

Treatment of Chironomus polytene chromosomes with the ultimate carcinogen benzo[a]pyrene diol epoxide I or in vivo administration of the parent hydrocarbon to larvae indicates that the carcinogen interacts with the genome in a nonrandom manner. Visualization of the carcinogen-DNA binding sites by immunofluorescence reveals that, in vivo, some sites are preferentially modified. The combined effects of DNA sequence, chromatin structure, and gene localization may lead to selective targeting of carcinogens to specific genomic regions. In polytene chromosomes the targeting effect is amplified, thereby making these chromosomes a uniquely suitable system for visualizing and studying site-specific interactions of carcinogens with the genome.     

Polymorphisms in the human haptoglobin gene cluster: chromosomes with multiple haptoglobin-related (Hpr) genes.

We have found polymorphisms for the number of tandemly arranged haptoglobin-related (Hpr) genes in the haptoglobin gene cluster of Blacks. Genomic mapping and nucleotide sequence analysis indicate that two copies of the Hpr gene first resulted from unequal but homologous crossing-over in a region 3' to the haptoglobin (Hp) and the haptoglobin-related genes. Subsequent increases in the number of Hpr loci have occurred in some chromosomes. Among 25 American Blacks studied (15 were unrelated), 2 related individuals have one extra copy of the Hpr gene and 5 unrelated individuals have more than two extra Hpr genes. None of 26 Whites and one Oriental studied have extra copies. In one of the Blacks, six tandemly arranged Hpr genes were demonstrated in one chromosome by pulsed field gradient electrophoresis. His other chromosome had one Hpr gene. The tandem Hpr genes were found in individuals with the haptoglobin genotypes Hp2/Hp2 (3 of 3 tested) and Hp2/Hp1 (4 of 11 tested), but none were found in the Hp1/Hp1 individuals (11 tested). Fibroblast cell cultures from two Hp2/Hp1 heterozygotes were fused to mouse cells to obtain cell lines retaining a human chromosome 16 on which the haptoglobin gene cluster is located. DNA analysis of the hybrid cells showed that in both individuals the tandemly arranged Hpr genes are linked to the Hp2 allele. These results suggest that the multiple copies are associated with the Hp2 gene.     

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-β and ori-γ) being somewhat preferred. We show here that the chromatin in the ori-β and ori-γ regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G₁/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.     

Transformation of Phycomyces blakesleeanus to G-418 resistance by an autonomously replicating plasmid.

The fungus, Phycomyces blakesleeanus, shows many well-defined responses to a number of external stimuli. Genetic analysis shows that at least eight genes are involved in Phycomyces sensory transduction. As a first step toward the molecular analysis of these genes and their products, we have developed a transformation protocol for Phycomyces by using a plasmid containing the kanamycin-resistance gene from Tn903 and a Phycomyces DNA fragment capable of supporting autonomous replication in yeast (ARS). Our results demonstrate that the Tn903 gene is expressed in Phycomyces and that the ARS fragment selected in yeast supports autonomous replication in Phycomyces as well.