Dosage Compensation Regulatory Proteins and the Evolution of Sex Chromosomes in Drosophila

In the fruitfly Drosophila melanogaster, the four male-specific lethal (msl) genes are required to achieve dosage compensation of the male X chromosome. The MSL proteins are thought to interact with cis-acting sites that confer dosage compensation to nearby genes, as they are detected at hundreds of discrete sites along the length of the polytene X chromosome in males but not in females. The histone H4 acetylated isoform, H4Ac16, colocalizes with the MSL proteins at a majority of sites on the D. melanogaster X chromosome. Using polytene chromosome immunostaining of other species from the genus Drosophila, we found that X chromosome association of MSL proteins and H4Ac16 is conserved despite differences in the sex chromosome karyotype between species. Our results support a model in which cis-acting regulatory sites for dosage compensation evolve on a neo-X chromosome arm in response to the degeneration of its former homologue.     

Heterochromatic chromosomes and satellite DNAs of Drosophila nasutoides

Drosophila nasutoides is distinguished from other Drosophila species in that the metaphase karyotype shows a pair of very large V-shaped chromosomes. With Giemsa, a distinctive C-banding pattern is revealed along the arms of this large chromosome, indicating a largely heterochromatic nature. Furthermore, the banding patterns of the arms are symmetrical, indicating that it is an iso-chromosome. A comparison between the metaphase karyotype and polytene chromosomes suggests that the large V chromosome appears as the dot chromosome in polytene squash. One autosome has twice the arm length of typical Drosophila polytene chromosomes and arose either by centric fusion and a pericentric inversion, or by translocation connecting distal ends with a subsequent loss of one centromere. This chromosome appears to have a short arm which ectopically pairs with the proximal region of the long arm, representing a duplication of about ten bands. When the nuclear DNA is examined by neutral CsCl gradient, four satellites are observed. As much as sixty percent of the total DNA appears as satellites in the lysate of larval brains. No satellite was detectable in the lysate of salivary glands. These observations led us to suggest that the heterochromatic nature of the large V chromosome is due to the presence of all four satellites in this chromosome and that this large chromosome appears as the dot because of the under-reduplication of the satellites during polytenization.     

The polytene dot chromosome of <Emphasis Type="Italic">Drosophila</Emphasis>: <Emphasis Type="Italic">D. melanogaster</Emphasis> and <Emphasis Type="Italic">D. subobscura</Emphasis>

The chromosome arms are assumed to be homologous within the genus Drosophila. Homology at the level of the polytene chromosome banding pattern between non-sibling species is, however, almost impossible to establish as different processes such as inversion, transposition and unequal crossing over, have disturbed it. Even though the band sequences cannot be followed, we may ask whether there is a correlation in the total number of bands between species. The polytene dot chromosome is an excellent starting point for such an approach. Here we present the detailed cytology of polytene chromosome 4 of D. melanogasterand the polytene dot chromosome of D. subobscura using electron microscopy. The results show that the number of bands is about the same, around 30, in both species. We predict that by using thin sections and electron microscopy for the longer polytene chromosome arms, both species will turn out to have approximately equal band numbers.     

Chromosomal homologies between Cebus and Ateles (primates) based on ZOO-FISH and G-banding comparisons

ZOO-FISH (Fluorescent "in vitro" hybridization) was used to establish the chromosomal homology between humans (HSA) and Cebus nigrivitatus (CNI) and Ateles belzebuth hybridus (ABH). These two species belong to different New World monkey families (Cebidae and Atelidae, respectively) which differ greatly in chromosome number and in chromosome morphology. The molecular results were followed by a detailed banding analysis. The ancestral karyotype of Cebus was then determined by a comparison of in situ hybridization results, as well as chromosomal morphology and banding in other Platyrrhini species. The karyotypes of the four species belonging to the genus Cebus differ from each other by three inversions and one fusion as well as in the location and amounts of heterochromatin. Results obtained by ZOO-FISH in ABH are in general agreement with previous gene-mapping and in situ hybridization data in Ateles, which show that spider monkeys have highly derived genomes. The chromosomal rearrangements detected between HSA and ABH on a band-to-band basis were 27 fusions/fissions, 12 centromeric shifts, and six pericentric inversions. The ancestral karyotype of Cebus was then compared with that of Ateles. The rearrangements detected were 20 fusions/fissions, nine centromeric shifts, and five inversions. Atelidae species are linked by a fragmentation of chromosome 4 into three segments forming an association of 4/15, while Ateles species are linked by 13 derived associations. The results also helped clarify the content of the ancestral platyrrhine karyotype and the mode of chromosomal evolution in these primates. In particular, associations 2/16 and 5/7 should be included in the ancestral karyotype of New World monkeys.     

Chromosomal Polymorphisms of Constitutive Heterochromatin and Inversions in Drosophila

A simple technique for preparing mitotic metaphases from a larval ganglion of Drosophila is described. Parallel examination of polytene and metaphase chromosome groups shows that inversion polymorphism in chromosome 3 of D. recticilia from East Maui (Hawaii) manifests a one-to-one correlation with a metaphase karyotype polymorphism due to the presence of an extra heterochromatic portion. These observations are consistent with the previous findings on other species of Hawaiian Drosophila. They strongly support the hypothesis that when one breakpoint of a long inverted segment of a chromosome element occurs in the vicinity of the constitutive heterochromatin, it may exert an effect in eliciting the production of heterochromatic material in the same chromosome.     

Karyotype studies of some species of Dalechampia Plum. (Euphorbiaceae)

Karyotype studies in eight species of Dalechampia , including 10 natural populations, revealed chromosome numbers (2 n = 36, 46, 138 and 198) differing from two numbers cited in the literature (2 n = 44 and 72). The basic number x = 6, as in the genus Acalypha , may be considered ancestral in Dalechampia. Analysis of chromosome number, haploid chromosome length and karyotype symmetry suggests that the major chromosome mechanism acting in karyotype evolution of Dalechampia is polyploidy, but differences in chromosome morphology may be caused by chromosome rearrangements.     

On the genomic location of the exuperantia1 gene in Drosophila miranda: the limits of in situ hybridization experiments

In situ hybridization to Drosophila polytene chromosomes is a powerful tool for determining the chromosomal location of genes. Using in situ hybridization experiments, Yi and Charlesworth recently reported the transposition of the exuperantia1 gene (exu1) from a neo-sex chromosome to the ancestral X chromosome of Drosophila miranda, close to exuperantia2 (exu2). By characterizing sequences flanking exu1, however, we found the position of exu1 to be conserved on the neo-sex chromosome. Further, the exu2 gene was found to be tandemly duplicated on the X chromosome of D. miranda. The misleading hybridization signal of exu1 may be caused by multiple copies of exu2, which interfere with the hybridization of the exu1 probe to its genomic position on the neo-X chromosome. This suggests that flanking DNA should be used to confirm the positions of members of gene families.     

Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps

The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.     

Patterns of replication in the neo-sex chromosomes ofDrosophila nasuta albomicans

Drosophila nasuta albomicans (with 2n = 6), contains a pair of metacentric neo-sex chromosomes. Phylogenetically these are products of centric fusion between ancestral sex (X, Y) chromosomes and an autosome (chromosome 3). The polytene chromosome complement of males with a neo-X- and neo-Y-chromosomes has revealed asynchrony in replication between the two arms of the neo-sex chromosomes. The arm which represents the ancestral X-chromosome is faster replicating than the arm which represents ancestral autosome. The latter arm of the neo-sex chromosome is synchronous with other autosomes of the complement. We conclude that one arm of the neo-X/Y is still mimicking the features of an autosome while the other arm has the features of a classical X/Y-chromosome. This X-autosome translocation differs from the other evolutionary X-autosome translocations known in certain species ofDrosophila.     

Genetic mapping of the Adh locus in the repleta group of Drosophila by in situ hybridization

A biotinylated probe of the Adh (alcohol dehydrogenase) gene of Drosophila melanogaster was used for in situ hybridization on polytene chromosomes of D. mojavensis and D. buzzatii, two species of the repleta group of the genus Drosophila. Hybridization showed that the Adh gene maps at the G1a band of the third chromosome. This is in accordance with a previous result obtained through the use of interspecific hybrid asynapsis as a cytological marker and establishes the limits of the precision of this method.     

A combined approach to mapping the proximal border of the right arm of polytene chromosome 2 in Drosophila melanogaster otu 11

A combined approach based on cytological observations in situ hybridization, and qualitative Southern-blot analyses were used to localize the proximal border of the right arm of polytene chromosome 2 in Drosophila melanogaster otu 11 strain. A genetically functional chromosome 2 is bounded by "deletions" C', C, D, B, A and ms2-10. Using in situ hybridization in conjunction with comparative quantitative Southern-blot hybridization to deletions in centromeric heterochromatin, DNA of specific centromeric clone lambda20p1.4 was localized with respect to "deletions" and on otu 11 polytene chromosomes. Comparison of hybridization sites of lambda20p1.4 on polytene chromosomes, and its amount in mutant lines of D. melanogaster carrying known "deletions" in the centromeric heterochromatin enabled us to localize the proximal border of the right arm of chromosome 2 in D. melanogaster otu 11 strain between the 39/40 region and hybridization site of the k20p1.4 DNA fragment.     

Cloning of a Drosophila melanogaster adenine phosphoribosyltransferase structural gene and deduced amino acid sequence of the enzyme

The Aprt locus of Drosophila melanogaster encodes the structural gene for adenine phosphoribosyltransferase (APRT). DNA cloned from microdissected salivary gland polytene chromosome region 62B7-12 was used in conjunction with chromosome walking and hybrid selection of mRNA to isolate the Aprt gene. Aprt lies at cytogenetic position 62B9 and is closely flanked by other genes of unknown function. Nucleotide sequencing shows that four APRT cDNAs have a common 5' terminus with an apparent cap consensus sequence but two different 3' sites of polyadenylation. The distribution of conserved amino acid sequences in APRT from vertebrates, insects and bacteria suggests that they may have shared a common ancestral gene for this ubiquitous enzyme.     

The Genetic and Cytogenetic Localization of the Three Structural Genes Coding for the Major Protein of Drosophila Larval Serum

The alpha, beta and gamma polypeptides that make up Drosophila Larval Serum Protein-1 seem to be coded for by genes that have evolved by duplication of a common ancestral gene. We have found variants of all three polypeptides, and these are variants of the coding sequences. The alpha-chain variant mapped to 39.5 on the X chromosome and to the polytene interval 11A7-11B9. The beta-chain variant mapped to 1.9 on chromosome 2L and to 21D2-22A1. The gamma-chain variant was mapped as 0.13 map units from the tip of chromosome 3L or to --1.41 with respect to ru, which has been defined as 0.0, and to 61A1-61A6.     

"Bar-coding" primate chromosomes: molecular cytogenetic screening for the ancestral hominoid karyotype

Two recently introduced multicolor FISH approaches, cross-species color banding (also termed Rx-FISH) and multiplex FISH using painting probes derived from somatic cell hybrids retaining fragments of human chromosomes, were applied in a comparative molecular cytogenetic study of higher primates. We analyzed these "chromosome bar code" patterns to obtain an overview of chromosomal rearrangements that occurred during higher primate evolution. The objective was to reconstruct the ancestral genome organization of hominoids using the macaque as outgroup species. Approximately 160 individual and discernible molecular cytogenetic markers were assigned in these species. Resulting comparative maps allowed us to identify numerous intra-chromosomal rearrangements, to discriminate them from previous contradicting chromosome banding interpretations and to propose an ancestral karyotype for hominoids. From 25 different chromosome forms in an ancestral karyotype for all hominoids of 2N=48 we propose 21. Probes for chromosomes 2p, 4, 9 and Y were not informative in the present experiments. The orangutan karyotype was very similar to the proposed ancestral organization and conserved 19 of the 21 ancestral forms; thus most chromosomes were already present in early hominoid evolution, while African apes and human show various derived changes.     

Reconstruction of ancestral karyotype illuminates chromosome evolution in the genus Cucumis

Karyotype dynamics driven by complex chromosome rearrangements constitute a fundamental issue in evolutionary genetics. The evolutionary events underlying karyotype diversity within plant genera, however, have rarely been reconstructed from a computed ancestral progenitor. Here, we developed a method to rapidly and accurately represent extant karyotypes with the genus, Cucumis, using highly customizable comparative oligo-painting (COP) allowing visualization of fine-scale genome structures of eight Cucumis species from both African-origin and Asian-origin clades. Based on COP data, an evolutionary framework containing a genus-level ancestral karyotype was reconstructed, allowing elucidation of the evolutionary events that account for the origin of these diverse genomes within Cucumis. Our results characterize the cryptic rearrangement hotspots on ancestral chromosomes, and demonstrate that the ancestral Cucumis karyotype (n = 12) evolved to extant Cucumis genomes by hybridizations and frequent lineage- and species-specific genome reshuffling. Relative to the African species, the Asian species, including melon (Cucumis melo, n = 12), Cucumis hystrix (n = 12) and cucumber (Cucumis sativus, n = 7), had highly shuffled genomes caused by large-scale inversions, centromere repositioning and chromothripsis-like rearrangement. The deduced reconstructed ancestral karyotype for the genus allowed us to propose evolutionary trajectories and specific events underlying the origin of these Cucumis species. Our findings highlight that the partitioned evolutionary plasticity of Cucumis karyotype is primarily located in the centromere-proximal regions marked by rearrangement hotspots, which can potentially serve as a reservoir for chromosome evolution due to their fragility.     

High-pressure treatment of polytene chromosomes improves structural resolution

The exceptional cytology provided by polytene chromosomes has made Drosophila melanogaster a premier model for chromosome studies, but full exploitation of polytene cytology is impeded by the difficulty in preparing high-quality chromosome spreads. Here we describe use of high pressure to produce formaldehyde-fixed chromosome spreads, which upon light-microscopy examination reveal structural detail previously observed only in electron microscopy preparations. We demonstrate applications to immunofluorescence and in situ hybridization.     

Chromosome evolution in kangaroos (Marsupialia: Macropodidae): cross species chromosome painting between the tammar wallaby and rock wallaby spp. with the 2n = 22 ancestral macropodid karyotype.

Marsupial mammals show extraordinary karyotype stability, with 2n = 14 considered ancestral. However, macropodid marsupials (kangaroos and wallabies) exhibit a considerable variety of karyotypes, with a hypothesised ancestral karyotype of 2n = 22. Speciation and karyotypic diversity in rock wallabies (Petrogale) is exceptional. We used cross species chromosome painting to examine the chromosome evolution between the tammar wallaby (2n = 16) and three 2n = 22 rock wallaby species groups with the putative ancestral karyotype. Hybridization of chromosome paints prepared from flow sorted chromosomes of the tammar wallaby to Petrogale spp., showed that this ancestral karyotype is largely conserved among 2n = 22 rock wallaby species, and confirmed the identity of ancestral chromosomes which fused to produce the bi-armed chromosomes of the 2n = 16 tammar wallaby. These results illustrate the fission-fusion process of karyotype evolution characteristic of the kangaroo group.     

Electron microscopic band-interband pattern of polytene chromosomes in Drosophila nasuta albomicans. 2. Salivary gland chromosome 2L.

The band-interband pattern (division 28-52) of salivary gland chromosome 2L in Drosophila nasuta albomicans was studied by light (LM) and electron microscopy (EM) using squash preparations and surface-spread polytene (SSP) chromosome preparations, respectively. LM and EM maps were complied. Based on the digitized EM patterns of five homologous SSP chromosomes a computerized EM chromosome map was plotted. The EM pattern analysis showed a total number of 479 chromosome bands with an almost 83% increase compared with the LM analysis of squash preparations. By extrapolation of the data from 39% of the polytene genome analysed so far in D. n. albomicans, a total number of 2,926 chromosome bands was calculated. This is almost the same number of bands as was calculated earlier for Drosophila hydei using the same SSP chromosome preparation technique. The data in the literature concerning variations in the number of chromosome bands in different Drosophila species, the various chromosome preparation techniques adopted, and the different criteria used for the EM pattern analyses, are discussed.     

开普芦荟和木立芦荟的染色体核型分析

对盆栽开普卢荟(Aloe ferox Miller)和木立卢荟(Aloe arborescens Miller)植物根尖细胞的染色体进行了观察分析。结果表明开普芦荟和木立芦荟的染色体数与已见报导的百合科(Liliaceae)中国芦荟(Alov vera var.chinensis)植物染色体数相同, 2n=14。染色体类型按Levan 方法分类, 没有近端部染色体和随体。开普芦荟和木立芦荟的染色体核型分析结果均为K(2n)=2x=4sm+10st。根据Stebbins 的核型分类标准, 开普芦荟的核型为"4C"型, 而木立芦荟的核型为"3C"型。两种芦荟染色体相对长度组成均为2n=14=6L+2M2+6S。根据核型研究, 可以确定百合科开普芦荟和木立芦荟的染色体基数为X=7。