Studies of Normal and Position-Affected Expression of ROSY Region Genes in DROSOPHILA MELANOGASTER

Transformant complementation, intragenic deletions and Northern blot analyses provide unambiguous localization of the l(3)S12 gene immediately proximal to the 5' end of the rosy locus. We have characterized an array of transformants with respect to l(3)S12 and rosy expression. The l(3)S12 gene is exceedingly sensitive to euchromatic site-specific position effects. Unlike the rosy locus, l(3)S12 is insensitive to heterochromatic position effect in rearrangements, as well as in a transformant located in heterochromatin. Cotransformants for both l(3)S12 and rosy elicit no apparent pattern of concordance with respect to euchromatic site-specific position effects. Heterochromatic-euchromatic rearrangements are examined with respect to position effects on expression of the rosy region genes l(3)12, rosy, snake and piccolo, as well as suppressor effects. Clear distinction is seen between euchromatic and heterochromatic effects.     

Genetic Limits of the Xanthine Dehydrogenase Structural Element within the Rosy Locus in DROSOPHILA MELANOGASTER

Experiments are described that provide an opportunity to estimate the genetic limits of the structural (amino acid coding) portion of the rosy locus (3:52.0) in Drosophila melanogaster, which controls the enzyme, xanthine dehydrogenase (XDH). This is accomplished by mapping experiments which localize sites responsible for electrophoretic variation in the enzyme on the known genetic map of null-XDH rosy mutants. Electrophoretic sites are distributed along a large portion of the null mutant map. A cis-trans test involving electrophoretic variants in the left- and right-hand portions of the map leads to the conclusion that the entire region between these variants is also structural. Hence most, if not all, of the null mutant map of the rosy locus contains structural information for the amino acid sequence of the XDH polypeptide. Consideration is given to the significance of the present results for the general problem of gene organization in higher eukaryotes.     

The Underlying Bases of Gene Expression Differences in Stable Transformants of the ROSY Locus in DROSOPHILA MELANOGASTER

This report represents a continuation of our laboratory's effort to understand the major phenomena associated with P-M dysgenesis-mediated transformation in Drosophila. A group of stable transformants are characterized with respect to rosy gene expression. Stable, true-breeding, line-specific variants in gene expression are described. These are shown to be associated with single transposons present in each line, and the lines are free of functional P elements. The effects on expression are cis-acting, and there are no identifiable rosy DNA sequence lesions associated with these transposons. Evidence is presented that demonstrates that two features of the transformation experimental system are responsible for such variation. The first relates to the fact that the transposons insert at numerous genomic sites. Both heterochromatic and euchromatic position effects are characterized. The second relates to the fact that transformation involves dysgenic mobilization of a P-element transposon. This process is mutagenic, and such a mutation is characterized.     

Heterochromatic Position Effect at the Rosy Locus of DROSOPHILA MELANOGASTER: Cytological, Genetic and Biochemical Characterization

This report describes cytological, genetic and biochemical studies designed to characterize two γ-radiation induced, apparent "underproducer" variants of the rosy locus (ry:3-52.0), ry^ps1149 and ry^ps11136. The following observations provide a compelling basis for their diagnosis as heterochromatic position effect variants. (1) They are associated with rearrangements that place heterochromatin adjacent to the rosy region of chromosome 3 (87D). (2) The effect of these mutations on rosy locus expression is subject to modification by abnormal Y chromosome content. (3) The rearrangement alters only the expression of the rosy allele on the same chromosome (cis-acting). (4) The Y chromosome modification is only on the position-affected allele's expression. (5) The recessive lethality associated with the rearrangements relate to specific rosy region vital loci, and for ry^{ps 11136}, the lethality is not Y chromosome modified. (6) The peptide product of the position-affected allele is qualitatively normal by several criteria. (7) Heterozygous deletion of 87E2-F2 is a suppressor of the rosy position effect. (8) The rosy position effect on XDH production may be assayed in whole larvae and larval fat body tissue as well as in adults.     

Dysgenesis-Induced Instability of Rosy Locus Transformation in DROSOPHILA MELANOGASTER: Analysis of Excision Events and the Selective Recovery of Control Element Deletions

Utilizing the method of P-M hybrid dysgenesis-mediated gene transfer to insert rosy locus DNA into various chromosomal locations, we recovered a transformed strain that carries an ry+ transposon inserted in or near the scalloped locus in polytene section 13F on the X chromosome. The resultant product, when stabilized, behaves as a homozygous and hemizygous viable and fertile extreme scalloped allele associated with wild-type expression of the rosy locus. We have labeled this allele, sdry+. This allele has been destabilized by subsequent P-M hybrid dysgenesis, and mutations were recovered that exhibit alterations in the rosy and/or scalloped phenotypes. Representative samples of all phenotypic classes have been characterized by Southern blot analyses of restricted DNA. The most common events are excisions of DNA wholly internal to the transposon and representing sections of rosy DNA. In addition to loss of rosy locus function, such excisions affect the scalloped locus expression.--A second dysgenesis experiment was carried out involving an ry+ transposon inserted in polytene section 16D on the X chromosome. A minimal estimate of the relative frequency of imprecise excisions, determined in this experiment is 75%.--A successful pilot experiment is described that utilizes dysgenic perturbation of the sdry+ allele to select for small deletions of the 5' noncoding region of the rosy locus.     

Molecular Mapping of the ROSY Locus in DROSOPHILA MELANOGASTER

The DNA from the chromosomal region of the Drosophila rosy locus has been examined in 83 rosy mutant strains. Several spontaneous and radiation-induced alleles were associated with insertions and deletions, respectively. The lesions are clustered in a 4-kb region. Some of the alleles identified on the DNA map have been located on the genetic map by fine-structure recombination experiments. The genetic and molecular maps are collinear, and the alignment identifies the DNA location of the rosy control region. A rosy RNA of 4.5 kb has been identified; its 5' end lies in or near the control region.     

Extension of the Limits of the Xdh Structural Element in DROSOPHILA MELANOGASTER

Experiments expanding the array of mutants affecting the xanthine dehydrogenase (XDH) structural element in Drosophila melanogaster are described. These include rosy eye color mutants which exhibit interallelic complementation, and mutants with normal eye color but lowered levels of XDH. Evidence is presented which argues that these are structural alterations in the enzyme. Recombination experiments were performed using these mutants as well as some electrophoretic variants. The two ends of the rosy locus are marked with mutant sites which are clearly structural in nature; the XDH structural element and the rosy null mutant map are completely concordant. A possible procedure to recover control element mutants is described.     

On the Identification of the ROSY Locus DNA in DROSOPHILA MELANOGASTER: Intragenic Recombination Mapping of Mutations Associated with Insertions and Deletions

DNA extracts of several rosy-mutation-bearing strains were associated with large insertions and deletions in a defined region of the molecular map believed to include the rosy locus DNA. Large-scale, intragenic mapping experiments were carried out that localized these mutations within the boundaries of the previously defined rosy locus structural element. Molecular characterization of the wild-type recombinants provides conclusive evidence that the rosy locus DNA is localized to the DNA segment marked by these lesions. One of the mutations, ry2101, arose from a P-M hybrid dysgenesis experiment and is associated with a copia insertion. Experiments are described which suggest that copia mobilizes in response to P-M hybrid dysgenesis. Relevance of the data to recombination in higher organisms is considered.     

Recombination Can Initiate and Terminate at a Large Number of Sites within the Rosy Locus of Drosophila Melanogaster

This report presents the results of a recombination experiment designed to question the existence of special sites for the initiation or termination of a recombination heteroduplex within the region of the rosy locus. Intragenic recombination events were monitored between two physically separated rosy mutant alleles ry301 and ry2 utilizing DNA restriction site polymorphisms as genetic markers. Both ry301 and ry2 are known from previous studies to be associated with gene conversion frequencies an order of magnitude lower than single site mutations. The mutations are associated with large, well defined insertions located as internal sites within the locus in prior intragenic mapping studies. On the molecular map, they represent large insertions approximately 2.7 kb apart in the second and third exons, respectively, of the XDH coding region. The present study monitors intragenic recombination in a mutant heterozygous genotype in which DNA homology is disrupted by these large discontinuities, greater than the region of DNA homology and flanking both sides of the locus. If initiation/or termination requires separate sites at either end of the locus, then intragenic recombination within the rosy locus of the heterozygote should be eliminated. Contrary to expectation, significant recombination between these sites is seen.     

Organization of the Rosy Locus in DROSOPHILA MELANOGASTER: Further Evidence in Support of a CIS-Acting Control Element Adjacent to the Xanthine Dehydrogenase Structural Element

The present report summarizes our recent progress in the genetic dissection of an elementary genetic unit in a higher organism, the rosy locus (ry:3--52.0) in Drosophila melanogaster. Pursuing the hypothesis that the rosy locus includes a noncoding control region, as well as a structural element coding for the xanthine dehydrogenase (XDH) peptide, experiments are described that characterize and map a rosy locus variant associated with much lower than normal levels of XDH activity. Experiments are described that fail to relate this phenotype to alteration in the structure of the XDH peptide, but clearly associate this character with variation in number of molecules of XDH per fly. Large-scale fine-structure recombination experiments locate the genetic basis for this variation in the number of molecules of XDH per fly to a site immediately to the left of the XDH structural element within a region previously designated as the XDH control element. Moreover, experiments clearly separate this "underproducer" variant site from a previously described "overproducer" site within the control region. Examination of enzyme activity in electrophoretic gels of appropriate heterozygous genotypes demonstrates the cis-acting nature of this variation in the number of molecules of XDH. A revision of the map of the rosy locus, structural and control elements is presented in the light of the additional mapping data now available.     

Cytogenetic Analysis of the Chromosomal Region Immediately Adjacent to the Rosy Locus in DROSOPHILA MELANOGASTER

This report describes the genetic analysis of a region of the third chromosome of Drosophila melanogaster extending from 87D2–4 to 87E12–F1, an interval of 23 or 24 polytene chromosome bands. This region includes the rosy (ry, 3–52.0) locus, carrying the structural information for xanthine dehydrogenase (XDH). We have, in recent years, focused attention on the genetic regulation of the rosy locus and, therefore, wished to ascertain in detail the immediate genetic environment of this locus. Specifically, we question if rosy is a solitary genetic unit or part of a larger complex genetic unit encompassing adjacent genes. Our data also provide opportunity to examine further the relationship between euchromatic gene distribution and polytene chromosome structure.——The results of our genetic dissection of the rosy microregion substantiate the conclusion drawn earlier (Schalet, Kernaghan and Chovnick 1964) that the rosy locus is the only gene in this region concerned with XDH activity and that all adjacent genetic units are functionally, as well as spatially, distinct from the rosy gene. Within the rosy micro-region, we observed a close correspondence between the number of complementation groups (21) and the number of polytene chromosome bands (23 or 24). Consideration of this latter observation in conjunction with those of similar studies of other chhromosomal regions supports the hypothesis that each polytene chromosome band corresponds to a single genetic unit.     

Analysis of Subtelomeric Heterochromatin in the Drosophila Minichromosome Dp1187 by Single P Element Insertional Mutagenesis

We investigated whether single P element insertional mutagenesis could be used to analyze heterochromatin within the Drosophila minichromosome Dp1187. Forty-five insertions of the P[lacZ,rosy+] element onto Dp1187 (recovered among 7,825 transpositions) were highly clustered. None was recovered in centromeric heterochromatin, but 39 occurred about 40 kb from the distal telomere within a 4.7-kb hotspot containing tandem copies of a novel 1.8-kb repetitive DNA sequence. The DNA within and distal to this region lacked essential genes and displayed several other properties characteristic of heterochromatin. The rosy+ genes within the inserted transposons were inhibited by position-effect variegation, and the subtelomeric region was underrepresented in polytene salivary gland cells. These experiments demonstrated that P elements preferentially transpose into a small subset of heterochromatic sites, providing a versatile method for studying the structure and function of these chromosome regions. This approach revealed that a Drosophila chromosome contains a large region of subtelomeric heterochromatin with specific structural and genetic properties.     

Replication and subnuclear location dynamics of the immunoglobulin heavy-chain locus in B-lineage cells

The murine immunoglobulin heavy-chain (Igh) locus provides an important model for understanding the replication of tissue-specific gene loci in mammalian cells. We have observed two DNA replication programs with dramatically different temporal replication patterns for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines and in ex vivo-expanded pro-B cells, the entire locus is replicated early in S phase. In three cell lines that exhibit the early-replication pattern, we found that replication forks progress in both directions through the constant-region genes, which is consistent with the activation of multiple initiation sites. In contrast, in plasma cell lines, replication of the Igh locus occurs through a triphasic pattern similar to that previously detected in MEL cells. Sequences downstream of the Igh-C alpha gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An approximately 500-kb transition region connecting sequences that replicate early and late is replicated progressively later in S. The formation of the transition region in different cell lines is independent of the sequences encompassed. In B-cell lines that exhibit a triphasic-replication pattern, replication forks progress in one direction through the examined constant-region genes. Timing data and the direction of replication fork movement indicate that replication of the transition region occurs by a single replication fork, as previously described for MEL cells. Associated with the contrasting replication programs are differences in the subnuclear locations of Igh loci. When the entire locus is replicated early in S, the Igh locus is located away from the nuclear periphery, but when Vh gene sequences replicate late and there is a temporal-transition region, the entire Igh locus is located near the nuclear periphery.     

Organization of the Rosy Locus in DROSOPHILA MELANOGASTER : Evidence for a Control Element Adjacent to the Xanthine Dehydrogenase Structural Element

From a collection of electrophoretic variants of XDH obtained from laboratory strains and natural populations, a stock was isolated that was associated with much greater than normal levels of XDH activity. Preliminary recombination experiments demonstrated that this character maps to the rosy locus. While a series of observations failed to relate this phenotype to alteration in the structure of the XDH polypeptide, kinetic and immunological experiments did succeed in associating this character with variation in number of molecules of XDH/fly. Large scale fine structure recombination experiments locate the genetic basis for this variation in number of molecules of XDH/fly to a site very close to, but definitely outside of, the genetic boundaries of the XDH structural information. Observations are described which eliminate the possibility that we are dealing with a tandem duplication of the XDH structural element. Turning to a regulatory role for this genetic element located adjacent to the XDH structural information, a simple experiment is described which demonstrates that it functions as a "cis-acting" regulator of the XDH structural element.     

Molecular Analysis of Recombination Events in Drosophila

The locations of crossover junctions and gene conversion tracts, isolated in the rosy gene of Drosophila melanogaster, were determined using DNA sequencing and denaturing gradient gel electrophoresis. Frequent DNA sequence polymorphisms between the parental genes served as unselected genetic markers. All conversion tracts were continuous, and half of the reciprocal crossover events had conversion tracts at the crossover junction. These experiments have also identified the sequence polymorphisms responsible for altered gene expression in two naturally occurring rosy variants.     

The Role of Heterochromatin in the Expression of a Heterochromatic Gene, the Rolled Locus of Drosophila Melanogaster

Constitutive heterochromatic regions of chromosomes are those that remain condensed through most or all of the cell cycle. In Drosophila melanogaster, the constitutive heterochromatic regions, located around the centromere, contain a number of gene loci, but at a much lower density than euchromatin. In the autosomal heterochromatin, the gene loci appear to be unique sequence genes interspersed among blocks of highly repeated sequences. Euchromatic genes do not function well when brought into the vicinity of heterochromatin (position-effect variegation). We test the possibility that the blocks of centromeric heterochromatin provide an environment essential for heterochromatic gene function. To assay directly the functional requirement of autosomal heterochromatic genes to reside in heterochromatin, the rolled (rl) gene, which is normally located deep in chromosome 2R heterochromatin, was relocated within small blocks of heterochromatin to a variety of euchromatic positions by successive series of chromosomal rearrangements. The function of the rl gene is severely affected in rearrangements in which the rl gene is isolated in a small block of heterochromatin, and these position effects can be reverted by rearrangements which bring the rl gene closer to any large block of autosomal or X chromosome heterochromatin. There is some evidence that five other 2R heterochromatic genes are also affected among these rearrangements. These findings demonstrate that the heterochromatic genes, in contrast to euchromatic genes whose function is inhibited by relocation to heterochromatin, require proximity to heterochromatin to function properly, and they argue strongly that a major function of the highly repeated satellite DNA, which comprises most of the heterochromatin, is to provide this heterochromatic environment.     

Direct FISH of 5S rDNA on tomato pachytene chromosomes places the gene at the heterochromatic knob immediately adjacent to the centromere of chromosome 1.

We describe a molecular cytogenetic procedure for high resolution physical mapping of DNA markers, an essential step toward construction of an integrated molecular-classical-cytological map. Tomato was selected as material because its pachytene chromosomes are amenable for study and because detailed molecular, classical, and cytological maps are available. Karyotyping of acetocarmine-stained pachytene chromosomes showing detailed cytogenetic landmarks was combined with direct FISH of the 5S rDNA gene. This enabled us to pinpoint the 5S rDNA gene to the first heterochromatic knob immediately adjacent to the centromere in the short arm of chromosome 1. Thus the position of the 5S rDNA gene on the molecular map was related to the position of the 5S rDNA on the cytogenetic map. The results also provide conclusive evidence of the location of a functional gene in the pericentric heterochromatic region, a rare event to date in plants. We conclude that karyotyping of pachytene chromosomes can be combined with FISH to map a DNA sequence to a cytogenetically defined region and to determine the chromatin origin of an expressed gene. Key words : direct fluorescence in situ hybridization, 5S rDNA, pachytene chromosomes, heterochromatic gene.