Cytogenetic analysis of the third chromosome heterochromatin of Drosophila melanogaster

Previous cytological analysis of heterochromatic rearrangements has yielded significant insight into the location and genetic organization of genes mapping to the heterochromatin of chromosomes X, Y, and 2 of Drosophila melanogaster. These studies have greatly facilitated our understanding of the genetic organization of heterochromatic genes. In contrast, the 12 essential genes known to exist within the mitotic heterochromatin of chromosome 3 have remained only imprecisely mapped. As a further step toward establishing a complete map of the heterochomatic genetic functions in Drosophila, we have characterized several rearrangements of chromosome 3 by using banding techniques at the level of mitotic chromosome. Most of the rearrangement breakpoints were located in the dull fluorescent regions h49, h51, and h58, suggesting that these regions correspond to heterochromatic hotspots for rearrangements. We were able to construct a detailed cytogenetic map of chromosome 3 heterochromatin that includes all of the known vital genes. At least 7 genes of the left arm (from l(3)80Fd to l(3)80Fj) map to segment h49-h51, while the most distal genes (from l(3)80Fa to l(3)80Fc) lie within the h47-h49 portion. The two right arm essential genes, l(3)81Fa and l(3)81Fb, are both located within the distal h58 segment. Intriguingly, a major part of chromosome 3 heterochromatin was found to be "empty," in that it did not contain either known genes or known satellite DNAs.     

1(2)41Aa,a heterochromatic gene of Drosophila melanogaster,is required for mitotic and meiotic chromosome condensation

Genetic and cytological approaches have yielded significant insight into the mapping and organization of genes located in the heterochromatin of Drosophila melanogaster. To date, only a few of these genes have been molecularly characterized in detail, and their function unveiled. As a further step towards the identification of heterochromatic gene functions, we have carried out a cytological analysis of mitotic and meiotic cell divisions in mutants carrying different allelic combinations of 1(2)41Aa, a gene located in the proximal heterochromatin of chromosome 2. Our results showed that larval brains of 1(2)41Aa mutants display a high frequency of cells with irregularly condensed chromosomes. In addition, defective chromosome condensation was detected in male meiosis, consequently affecting chromosome segregation and giving rise to irregular spermatids. Taken together, these findings indicate that 1(2)41Aa is a novel cell cycle gene required for proper chromosome condensation in both somatic and germ line cells.     

The Effects of Chromosome Rearrangements on the Expression of Heterochromatic Genes in Chromosome 2l of Drosophila Melanogaster

The light (lt) gene of Drosophila melanogaster is located at the base of the left arm of chromosome 2, within or very near centromeric heterochromatin (2Lh). Chromosome rearrangements that move the lt+ gene from its normal proximal position and place the gene in distal euchromatin result in mosaic or variegated expression of the gene. The cytogenetic and genetic properties of 17 lt-variegated rearrangements are described in this report. We show that five of the heterochromatic genes adjacent to lt are subject to inactivation by these rearrangements and that the euchromatic loci in proximal 2L are not detectably affected. The properties of the rearrangements suggest that proximity to heterochromatin is an important regulatory requirement for at least six 2Lh genes. We discuss how the properties of the position effects on heterochromatic genes relate to other proximity-dependent phenomena such as transvection.     

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.     

Genetic Analysis of the Centromeric Heterochromatin of Chromosome 2 of DROSOPHILA MELANOGASTER: Deficiency Mapping of Ems-Induced Lethal Complementation Groups

Until recently, little was known of the genetic constitution of the heterochromatic segments of the major autosomes of Drosophila melanogaster . Our previous report described the genetic dissection of the proximal, heterochromatic region of chromosome 2 of Drosophila melanogaster by means of a series of overlapping deficiencies generated by the detachment of compound second autosomes (Hilliker and Holm 1975). Analysis of these deficiencies by inter se complementation, pseudo-dominance tests with proximal mutations and allelism tests with known deficiencies provided evidence for the existence of at least two loci between the centromere and the light locus in 2L and one locus in 2R between the rolled locus and the centromere. These data in conjunction with cytological observations demonstrated that light and rolled and three loci lying between them are located within the proximal heterochromatin of the second chromosome.——The present report describes the further analysis of this region through the induction with ethyl methanesulphonate (EMS) of recessive lethals allelic to the 2L and 2R proximal deficiencies associated with the detachment products. Analysis of the 118 EMS-induced recessive lethals and visible mutations recovered provided evidence for seven loci in the 2L heterochromatin and six loci in the 2R heterochromatin, with multiple alleles being obtained for most sites. Of these loci, one in 2L and two in 2R fall near the heterochromatic-euchromatic junctions of 2L and 2R respectively. None of the 113 EMS lethals behaved as a deficiency, implying that the heterochromatic loci uncovered in this study represent nonrepetitive cistrons. Thus functional genetic loci are found in heterochromatin, albeit at a very low density relative to euchromatin.     

A strategy for mapping the heterochromatin of chromosome 2 of Drosophila melanogaster

The heterochromatin of chromosome 2 of Drosophila melanogaster has been among the best characterized models for functional studies of heterochromatin owing to its abundance of genetic markers. To determine whether it might also provide a favorable system for mapping extended regions of heterochromatin, we undertook a project to molecularly map the heterochromatin of the left arm of chromosome 2 (2Lh). In this paper, we describe a strategy that used clones and sequence information available from the Drosophila Genome Project and chromosome rearrangements to construct a map of the distal most portion of 2Lh. We also describe studies that used fluorescent in situ hybridization (FISH) to examine the resolution of this technique for cytologically resolving heterochromatic sequences on mitotic chromosomes. We discuss how these mapping studies can be extended to more proximal regions of the heterochromatin to determine the structural patterns and physical dimensions of 2Lh and the relationship of structure to function.     

Integrated cytogenetic map of chromosome arm 4S of A. thaliana: structural organization of heterochromatic knob and centromere region

We have constructed an integrated cytogenetic map of chromosome arm 4S of Arabidopsis thaliana. The map shows the detailed positions of various multicopy and unique sequences relative to euchromatin and heterochromatin segments. A quantitative analysis of the map positions at subsequent meiotic stages revealed a striking pattern of spatial and temporal variation in chromatin condensation for euchromatin and heterochromatin. For example, the centromere region consists of three domains with distinguishable structural, molecular, and functional properties. We also characterized a conspicuous heterochromatic knob of approximately 700 kb that accommodates a tandem repeat and several dispersed pericentromere-specific repeats. Moreover, our data provide evidence for an inversion event that relocated pericentromeric sequences to an interstitial position, resulting in the heterochromatic knob.     

Alpha and beta heterochromatin in polytene chromosome 2 ofDrosophila melanogaster

The formation of alpha and beta heterochromatin in chromosomes of Drosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs of X0, XY, XYY, XX and XXY individuals the amounts of alpha heterochromatin were similar, suggesting that the Y chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization with Rsp sequences (H _o clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed. Received: 17 November 1995; in revised form: 10 April 1996 / Accepted: 18 September 1996     

Mapping of a single locus capable of complementing the defective heterochromatin phenotype of Roberts syndrome cells

Roberts syndrome (RS) is a developmental disorder characterized by tetraphocomelia and a broad spectrum of additional clinical features. Most patients with RS exhibit characteristic cytogenetic phenotypes, which include an abnormal appearance of pericentromeric heterochromatin on metaphase chromosomes, referred to as "heterochromatic repulsion." In the present study, we use complementation of this abnormal cytogenetic phenotype as a means to identify a specific region of the normal human genome capable of rendering phenotypic correction. We screened the entire human genome, using a transient chromosome-transfer assay, and demonstrated complementation exclusively after the transfer of proximal chromosome 8p, a result subsequently confirmed by stable microcell-mediated chromosome transfer. Additionally, homozygosity mapping was used to refine the interval of this complementing locus to 8p21. The results are consistent with the notion that the single gene defect responsible for heterochromatic splaying and developmental abnormalities maps to chromosome 8p21.     

Cytogenetic Analysis of Segregation Distortion in Drosophila Melanogaster: The Cytological Organization of the Responder (Rsp) Locus

The segregation distortion phenomenon occurs in Drosophila melanogaster males carrying an SD second chromosome and an SD+ homolog. In such males the SD chromosome is transmitted to the progeny more frequently than the expected 50% because of an abnormal differentiation of the SD+-bearing sperms. Three major loci are involved in this phenomenon: SD and Rsp, associated with the SD and SD+ chromosome, respectively, and E(SD). In the present work we performed a cytogenetic analysis of the Rsp locus which was known to map to the centromeric heterochromatin of the second chromosome. Hoechst- and N-banding techniques were used to characterize chromosomes carrying Responder insensitive (Rspi), Responder sensitive (Rsps) and Responder supersensitive (Rspss) alleles. Our results locate the Rsp locus to the h39 region of 2R heterochromatin. This region is a Hoechst-bright, N-banding negative heterochromatic block adjacent to the centromere. Quantitative variations of the h39 region were observed. The degree of sensitivity to Sd was found to be directly correlated with the physical size of that region, demonstrating that the Rsp locus is composed of repeated DNA.     

白鱀豚的核型及其C－带核型的研究

采用常规的白细胞培养技术及ＢＳＧＣ－带技术分析了白豚的核型、Ｃ－带核型。结果是：白豚的染色体数目２ｎ＝４４,其核型由１２条中部着丝点染色体、１８条亚中部着丝点染色体、４条亚端部着丝点染色体、８条端部着丝点染色体和２条性染色体所组成。染色体臂数（ＮＦ）雌性为７６,雄性为７５。Ｃ－带异染色质呈现出很深的着色区,主要分布在染色体臂上,着丝点区则几乎没有。Ｃ－带异染色质的量约为总染色质量的１２％。这一结果表明白豚与海生豚类的核型、Ｃ－带核型有较明显的相似性。     

The Heterochromatic Rolled Gene of Drosophila Melanogaster Is Extensively Polytenized and Transcriptionally Active in the Salivary Gland Chromocenter

This paper reports a cytogenetic and molecular study of the structural and functional organization of the Drosophila melanogaster chromocenter. The relations between mitotic (constitutive) heterochromatin and α- and β-heterochromatin are not fully understood. In the present work, we have studied the polytenization of the rolled (rl) locus, a 100-kb genomic region that maps to the proximal heterochromatin of chromosome 2 and has been previously thought to contribute to α-heterochromatin. We show that rolled undergoes polytenization in salivary gland chromosomes to a degree comparable to that of euchromatic genes, despite its deep heterochromatic location. In contrast, both the Bari-1 sequences and the AAGAC satellite repeats, located respectively to the left and right of rl, are severely underrepresented and thus both appear to be α-heterochromatic. In addition, we found that rl is transcribed in polytene tissues. Together, the results reported here indicate that functional sequences located within the proximal constitutive heterochromatin can undergo polytenization, contributing to the formation of β-heterochromatin. The implications of this finding to chromocenter structure are discussed.     

Unusual primitive heteromorphic ZZ/ZW sex chromosomes in Proceratophrys boiei (Anura, Cycloramphidae, Alsodinae), with description of C-Band interpopulational polymorphism

We performed cytogenetic analyses on specimens from three population samples of Proceratophrys boiei from southeastern and northeastern Brazil. We stained chromosomes of mitotic and meiotic cells with Giemsa, C-banding and Ag-NOR methods. All specimens of P. boiei presented a karyotype with a full chromosome complement of 2n=22, metacentric and submetacentric. We observed the secondary constriction within the short arm of pair 8, which was in the same position of the nucleolus organizer region (NOR). NOR heteromorphism was observed within two specimens from the municipality of Mata de S?o Jo?o (northeastern Bahia State). The C-banding evidenced an unusual heterochromatic pattern in the genome of P. boiei. In the southern most population samples (S?o Paulo State), we observed large blocks of heterochromatin in the centromeric regions of all chromosomes, whereas the northernmost samples (Bahia State) presented a small amount of constitutive heterochromatin. We suppose that this geographic variation in heterochromatin quantities could be due to heterochromatinization of some chromosome regions in the genome of the S?o Paulo samples. Furthermore, females from S?o Paulo presented, within chromosome pair 1 from C-banded karyotypes, one homologous chromosome almost heterochromatic, whereas males had heterochromatin restricted to the centromeric region. This unusual heterochromatic arrangement led us to assume that P. boiei owns a ZZ/ZW type of sexual determination system. This finding is very important, as this is the first record of ZZ/ZW sex chromosomes within Cycloramphidae. We believe that the cytogenetic differences found between southeastern and northeastern Brazilian population samples of P. boiei strongly supports the existence of a species complex under the name P. boiei, and the requirement of taxonomic and systematic reviews by morphological, bioacoustical, molecular, and cytogenetic data could define this taxonomic issue in the future.     

Genetic Analysis of the Proximal Region of Chromosome 2 of DROSOPHILA MELANOGASTER. I. Detachment Products of Compound Autosomes

To examine the genetic composition of proximal heterochromain in chromosome 2, the detachment of compound second autosomes, for generating proximal deficiencies, appeared a promising method. Compound seconds were detached by gamma radiation. A fraction of the detachment products were recessive lethals owing to proximal deficiencies. Analysis by inter se complementation, pseudo-dominance tests with proximal mutations and allelism tests with known deficiencies provided evidence for at least two loci between the centromere and the light locus in 2L and one locus in 2R between the rolled locus and the centromere. The data further demonstrate that rolled, and probably light, are located within the proximal heterochromatin. Thus, functional genetic loci are found in heterochromatin, albeit at low density.     

Studies on the linkage map of chromosome 4 of the tomato and on the transmission of induced deficiencies

Various genetic and cytogenetic techniques were applied to an analysis of the linkage map of chromosome 4-a chromosome that is considered to be representative of the tomato complement. Loci have been approximated by standard F₂ linkage tests for 18 genes, including six on the short arm and 12 on the long arm, covering a map distance of 132 units (c.m.). The loci of four key markers were approximated on pachytene chromosomes by a study of radiation-induced deficiencies:clau near the end of the short arm,ful near the euchromatic-heterochromatic boundary of the short arm,ra near the same region on the long arm, ande in the middle of the long arm. Normal transmission for a presumedra deficiency suggests that this gene lies in the heterochromatin of 4L. According to tertiary trisomic segregation,w-4, known by linkage test to be proximal tora, resides on 4L, therefore probably also in the heterochromatic region. The centromere is consequently delimited to a region of 4 c.m. betweenful andw-4. The resultant maps reveal a very much lower crossover rate within heterochromatin—estimated at 0.8 c.m./μ—than for euchromatin—estimated at 4.8 c.m./μ for the short arm and 5.7 for the long arm. Also apparent is a strong tendency of the genes to concentrate toward the centromere of the genetic map and in the proximal sections of the euchromatin of the cytological map. Studies were made of the genetic transmission of various small deficiencies on chromosome 4 as well as a newly discovered deficiency fornv on chromosome 9, supporting the following conclusions. Regardless of their size, deficiendies of euchromatin are not transmitted. Deficiencies of heterochromatin are transmitted to a varying extent depending on their size. A presumed deficiency forra that is too small to be detected cytologically was transmitted without adverse effect on gametes. Somewhat larger deficiencies may be transmitted at reduced rates by female gametes and the largest at extremely low rates, even on the female side.     

Meiotic effects of supernumerary heterochromatin in Heteropternis obscurella

The endemic Australian grasshopper Heteropternis obscurella shows considerable variation in respect of both chromosome structure and chromosome behaviour. The structural differences depend upon different patterns of heterochromatin distribution as revealed by C-banding. These involve differences between populations in respect of polytypic variation in the size of paracentromeric C-bands and differences within populations in respect of polymorphisms both for terminal blocks of heterochromatin in autosomes 3 to 8 and a large proximal block of heterochromatin in autosome 7. The behavioural differences stem in part from genotypically determined variation in the chiasma distribution pattern which is markedly localised in a majority of populations but more randomly distributed in populations from the south of Western Australia. Behavioural differences also arise as secondary consequences of the presence of those heterochromatic blocks which occur as polymorphisms. The distal blocks on autosomes 5, 6, 7 and 8 lead to a redistribution of chiasmata to more proximal sites while the proximal block on 7 leads to the virtual abolition of chiasma formation in that bivalent and its replacement by a non-chiasmate mechanism of segregation. This depends upon a persistent proximal heterochromatic association between the pairing partners. The presence of distal C-blocks on bivalents 3 to 8 gives rise to persistent pseudomultiples, formed as a result of heterochromatic associations between these blocks. Such pseudomultiples involve any two or three of these six bivalents, provided they carry distal blocks, and their frequency rises dramatically in the presence of the proximal heterochromatic block on chromosome 7.     

Genetic analysis of RpL38 and RpL5, two minute genes located in the centric heterochromatin of chromosome 2 of Drosophila melanogaster

The Minute mutations of Drosophila melanogaster are thought to disrupt genes that encode ribosomal proteins (RPs) and thus impair ribosome function and protein synthesis. However, relatively few Minutes have been tied to distinct RP genes and more Minute loci are likely to be discovered. We have identified point mutations in RpL38 and RpL5 in a screen for factors limiting for growth of the D. melanogaster wing. Here, we present the first genetic characterization of these loci. RpL38 is located in the centric heterochromatin of chromosome arm 2R and is identical to a previously identified Minute, M(2)41A, and also l(2)41Af. RpL5 is located in the 2L centric heterochromatin and defines a novel Minute gene. Both genes are haplo-insufficient, as heterozygous mutations cause the classic Minute phenotypes of small bristles and delayed development. Surprisingly, we find that RpL38(-)/+ and RpL5(-)/+ adult flies have abnormally large wings as a result of increased cell size, emphasizing the importance of translational regulation in the control of growth. Taken together, our data provide new molecular and genetic information on two previously uncharacterized Minute/RP genes, the heterochromatic regions in which they reside, and the role of their protein products in the control of organ growth.     

FISH analysis of<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> heterochromatin using BACs and<Emphasis Type="Italic"> P</Emphasis> elements

The heterochromatin of chromosomes 2 and 3 of Drosophila melanogaster contains about 30 essential genes defined by genetic analysis. In the last decade only a few of these genes have been molecularly characterized and found to correspond to protein-coding genes involved in important cellular functions. Moreover, several predicted genes have been identified by annotation of genomic sequence that are associated with polytene chromosome divisions 40, 41 and 80 but their locations on the cytogenetic map of the heterochromatin are still uncertain. To expand our current knowledge of the genetic functions located in heterochromatin, we have performed fluorescence in situ hybridization (FISH) mapping to mitotic chromosomes of nine bacterial artificial chromosomes (BACs) carrying several predicted genes and of 13 P element insertions assigned to the proximal regions of 2R and 3L. We found that 22 predicted genes map to the h46 region of 2R and eight map to the h47 regions of 3L. This amounts to at least 30 predicted genes located in these heterochromatic regions, whereas previous studies detected only seven vital genes. Finally, another 58 genes localize either in the euchromatin-heterochromatin transition regions or in the proximal euchromatin of 2R and 3L. Edited by: B. McKeeN. Corradini and F. Rossi contributed equally to this work     

Electron microscope mapping of the pericentric and intercalary heterochromatic regions of the polytene chromosomes of the mutant Suppressor of underreplication in Drosophila melanogaster

Breaks and ectopic contacts in the heterochromatic regions of Drosophila melanogaster polytene chromosomes are the manifestations of the cytological effects of DNA underreplication. Their appearance makes these regions difficult to map. The Su(UR)ES gene, which controls the phenomenon, has been described recently. Mutation of this locus gives rise to new blocks of material in the pericentric heterochromatic regions and causes the disappearance of breaks and ectopic contacts in the intercalary heterochromatic regions, thereby making the banding pattern distinct and providing better opportunities for mapping of the heterochromatic regions in polytene chromosomes. Here, we present the results of an electron microscope study of the heterochromatic regions. In the wild-type salivary glands, the pericentric regions correspond to the beta-heterochromatin and do not show the banding pattern. The most conspicuous cytological effect of the Su(UR)ES mutation is the formation of a large banded chromosome fragment comprising at least 25 bands at the site where the 3L and 3R proximal arms connect. In the other pericentric regions, 20CF, 40BF and 41BC, 15, 12 and 9 new bands were revealed, respectively. A large block of densely packed material appears in the most proximal part of the fourth chromosome. An electron microscope analysis of 26 polytene chromosome regions showing the characteristic features of intercalary heterochromatin was also performed. Suppression of DNA underreplication in the mutant transforms the bands with weak spots into large single bands.     

Genetic Analysis of the Heterochromatin of Chromosome 3 in Drosophila Melanogaster. II. Vital Loci Identified through Ems Mutagenesis

Chromosome 3 of Drosophila melanogaster contains the last major blocks of heterochromatin in this species to be genetically analyzed. Deficiencies of heterochromatin generated through the detachment of compound-3 chromosomes revealed the presence of vital loci in the heterochromatin of chromosome 3, but an extensive complementation analysis with various combinations of lethal and nonlethal detachment products gave no evidence of tandemly repeated vital genes in this region. These findings indicate that the heterochromatin of chromosome 3 is genetically similar to that of chromosome 2. A more thorough genetic analysis of the heterochromatic regions has been carried out using the chemical mutagen ethyl methanesulfonate (EMS). Seventy-five EMS-induced lethals allelic to loci uncovered by detachment-product deficiencies were recovered and tested for complementation. In total, 12 complementation groups were identified, ten in the heterochromatin to the left of the centromere and two to the right. All but two complementation groups in the left heterochromatic block could be identified as separate loci through deficiency mapping. The interallelic complementation observed between some EMS-induced lethals, as well as the recovery of a temperature-sensitive allele for each of the two loci, provided further evidence that single-copy, transcribed vital genes reside in the heterochromatin of chromosome 3. Cytological analysis of three detachment-product deficiencies provided evidence that at least some of the genes uncovered in this study are located in the most distal segments of the heterochromatin in both arms. This study provides a detailed genetic analysis of chromosome 3 heterochromatin and offers further information on the genetic nature and heterogeneity of Drosophila heterochromatin.