Histone H1 Genes and Histone Gene Clusters in the Genus Drosophila

Whereas the genomes of many organisms contain several nonallelic types of linker histone genes, one single histone H1 type is known in Drosophila melanogaster that occurs in about 100 copies per genome. Amplification of H1 gene sequences from genomic DNA of wild type strains of D. melanogaster from Oregon, Australia, and central Africa yielded numerous clones that all exhibited restriction patterns identical to each other and to those of the known H1 gene sequence. Nucleotide sequences encoding the evolutionarily variable domains of H1 were determined in two gene copies of strain Niamey from central Africa and were found to be identical to the known H1 sequence. Most likely therefore, the translated sequences of D. melanogaster H1 genes do not exhibit intragenomic or intergenomic variations. In contrast, three different histone H1 genes were isolated from D. virilis and found to encode proteins that differ remarkably from each other and from the H1 of D. melanogaster and D. hydei. About 40 copies of H1 genes are organized in the D. virilis genome with copies of core histone genes in gene quintets that were found to be located in band 25F of chromosome 2. Another type of histone gene cluster is present in about 15 copies per genome and contains a variable intergenic sequence instead of an H1 gene. The H1 heterogeneity in D. virilis may have arisen from higher recombination rates than occur near the H1 locus in D. melanogaster and might provide a basis for formation of different chromatin subtypes. Received: 2 March 2000 / Accepted: 1 June 2000     

Drosophila virilis histone gene clusters lacking H1 coding segments

Summary Approximately 30–40% ofDrosophila virilis DNA complementary to clonedDrosophila histone genes is reduced to 3.4-kilobase-pair (kbp) segments by Bgl I or Bgl II digestion. The core histone genes of a 3.4-kbp Bgl II segment cloned in the plasmid pDv3/3.4 have the same order as theD. melanogaster core histone genes in the plasmid cDm500: . Nonetheless, pDv3/3.4 and cDm500 have different histone gene configurations: In pDv3/3.4, the region between the H2B and H3 genes contains 0.35 kbp and cannot encode histone H1; in cDm500, the region contains 2.0 kbp and encodes histone H1. The lack of an H1 gene between the H2B and H3 genes in 30–40% ofD. virilis histone gene clusters suggests that changes in histone gene arrays have occurred during the evolution ofDrosophila. The ancestors of modernDrosophila may have possessed multiple varieties of histone gene clusters, which were subsequently lost differentially in thevirilis andmelanogaster lineages. Alternatively, they may have possessed a single variety, which was rearranged during evolution. The H1 genes ofD. virilis andD. melanogaster did not cross-hybridize in vitro under conditions that maintain stable duplexes between DNAs that are 75% homologous. Consequently,D. virilis H1 genes could not be visualized by hybridization to an H1-specific probe and thus remain unidentified. Our observations suggest that the coding segments in the H1 genes ofD. virilis andD. melanogaster are >25% divergent. Our estimate of sequence divergence in the H1 genes ofD. virilis andD. melanogaster seems high until one considers that the coding sequences of cloned H1 genes from the closely related speciesD. melanogaster andD. simulans are 5% divergent.     

Nucleotide and repeat length variation at the nonA gene of the Drosophila virilis group species and its effects on male courtship song

Genes found to affect male courtship song characters in Drosophila melanogaster are good candidates when tracing genes responsible for species-specific songs in other Drosophila species. It has previously been shown that Thr–Gly repeat length variation at the period gene affects song traits in D. melanogaster, which gives the repetitive regions a special interest. In this work, we have characterised the patterns of nucleotide variation for gene regions containing two Gly and one Gln–Ala repeat in another D. melanogaster song gene, no-on-transient A, in D. virilis group species. The levels of nucleotide variability in D. virilis nonA were similar to those found for other genes of the species, and the gene sequences showed no signs of deviation from neutrality. The Gly 2 repeat preceding the central domain of the gene exhibited length variation, which did not, however, correlate with song variation either within D. virilis or between the species of D. virilis group. The Gly 3 repeat located on the other side of the central domain showed amino acid divergence parallel to the consensus phylogeny of the D. virilis group species. The species of the virilis subgroup having Asn after the first three glycines in this repeat have simple songs with no species-specificity, while the species of the montana subgroup having two Gly or Asn–Ser in this site have unique courtship songs. Amino acid differences between the species in this repeat may, however, reflect species phylogeny rather than have an effect on song divergence per se.     

Comparison of dot chromosome sequences from D. melanogaster and D. virilis reveals an enrichment of DNA transposon sequences in heterochromatic domains

Background

Chromosome four of Drosophila melanogaster, known as the dot chromosome, is largely heterochromatic, as shown by immunofluorescent staining with antibodies to heterochromatin protein 1 (HP1) and histone H3K9me. In contrast, the absence of HP1 and H3K9me from the dot chromosome in D. virilis suggests that this region is euchromatic. D. virilis diverged from D. melanogaster 40 to 60 million years ago.

Results

Here we describe finished sequencing and analysis of 11 fosmids hybridizing to the dot chromosome of D. virilis (372,650 base-pairs) and seven fosmids from major euchromatic chromosome arms (273,110 base-pairs). Most genes from the dot chromosome of D. melanogaster remain on the dot chromosome in D. virilis, but many inversions have occurred. The dot chromosomes of both species are similar to the major chromosome arms in gene density and coding density, but the dot chromosome genes of both species have larger introns. The D. virilis dot chromosome fosmids have a high repeat density (22.8%), similar to homologous regions of D. melanogaster (26.5%). There are, however, major differences in the representation of repetitive elements. Remnants of DNA transposons make up only 6.3% of the D. virilis dot chromosome fosmids, but 18.4% of the homologous regions from D. melanogaster; DINE-1 and 1360 elements are particularly enriched in D. melanogaster. Euchromatic domains on the major chromosomes in both species have very few DNA transposons (less than 0.4 %).

Conclusion

Combining these results with recent findings about RNAi, we suggest that specific repetitive elements, as well as density, play a role in determining higher-order chromatin packaging.     

<Emphasis Type="Italic">Mytilus edulis</Emphasis> Core Histone Genes Are Organized in Two Clusters Devoid of Linker Histone Genes

Abstract Comparison of histone gene cluster arrangements in several species has revealed a broad spectrum of histone gene patterns. To elucidate the core histone gene organization in a mollusk, we have analyzed a Mytilus edulis genomic library and have isolated eight phage clones containing core histone genes. Analysis of insert DNA revealed that the core histone genes are arranged as regular gene repeats of all four core histones. The repeats do not contain linker histone genes. The clones are distributed into two groups of dissimilar repeated units with a common size of about 5.6 kb. The genes of each core histone class in the distinct repeats encode identical histone proteins and have comparable gene arrangements in the two repeat units. However, the intergenic sequences differ significantly. The core histone genes are organized as large clusters of about 100 repeats each. Previously, we have shown that the linker histone genes in M. edulis are also organized in a cluster of repeats of solitary H1 genes. Hence, this is the first case of a separate, clustered organization of both core and linker histone genes, repectively.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Evolution of 5S ribosomal RNA genes in the chromosomes of the virilis group of Drosophila

Drosophila melanogaster 5S ribosomal RNA labeled with ¹²⁵I was used as an in situ hybridization probe to localize complementary sequences in chromosomes of species in the Drosophila virilis group. Whereas virilis, the ancestral species, has two different 5S gene loci, the derived species show only one of these loci; in the two lines that have evolved from virilis it is the opposite locus that is conserved. The possible events leading to such an arrangement are discussed.The author was a Predoctoral Fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.Contribution from Oak Ridge National Laboratory, operated by the Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

Structural and functional analysis of a new retrotransposon class in Drosophila species

Several copies of the Penelope transposable element, previously described in Drosophila virilis, have been studied in different D. virilis strains and D. melanogaster strains transformed with P-based constructs bearing a full-size Penelope copy. Most Penelope copies in both species have large terminal inverted repeats (TIRs) and deletions of various sizes at the 5′ ends of their ORFs. Junctions between TIRs and ORFs usually have microhomologies of various lengths, which allowed a hypothesis explaining the emergence of these complex structures at the molecular level to be put forward. Most Penelope copies have a 34 bp long direct repeat at the ORF ends. Full-size and truncated Penelope copies are usually surrounded by target site duplications of various lengths.     

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster

Summary Previous studies have demonstrated that the expression of the -amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the -amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.Offprint requests to: D.A. Hickey     

Evolutionary divergence of the cytochrome b5 gene of Drosophila

Cytochrome proteins perform a broad spectrum of biological functions ranging from oxidative metabolism to electron transport and are thus essential to all organisms. The b-type cytochrome proteins bind heme noncovalently, are expressed in many different forms and are localized to various cellular compartments. We report the characterization of the cytochrome b₅ (Cyt-b) gene of Drosophila virilis and compare its structure to the Cyt-b gene of Drosophila melanogaster. As in D. melanogaster, the D. virilis gene is nuclear encoded and single copy. Although the intron/exon structures of these homologues differ, the Cyt-b proteins of D. melanogaster and D. virilis are approximately 75% identical and share the same size coding regions (1,242 nucleotides) and protein products (414 amino acids). The Drosophila Cyt-b proteins show sequence similarity to other b-type cytochromes, especially in the N-terminal heme-binding domain, and may be targeted to the mitochondrial membrane. The greatest levels of similarity are observed in areas of potential importance for protein structure and function. The exon sequences of the D. virilis Cyt-b gene differ by a total of 292 base changes. However, 62% of these changes are silent. The high degree of conservation between species separated by 60 million years of evolution in both the DNA and amino acid sequences suggests this nuclear cytochrome b₅ locus encodes an essential product of the Drosophila system.Correspondence to: C.E. Rozek     

Histone content in relation to amount of heterochromatin and developmental stage in three species of Drosophila

Relative amounts of various histone fractions in Drosophila chromatin were estimated densitometrically on electrophoretic gel separations. Several consistent and highly significant differences were obtained between larval and adult chromatin. The arginine-rich histones showed the most conspicuous changes: higher amounts of H4 in larvae, higher H3 in adults. The level of modification of these histones was clearly higher in larval than in adult chromatin. The modification of the two slower subfractions of H4 involved, in all probability, phosphorylation as well as acetylation. In all types of Drosophila chromatin studied 50% or more of the H2a molecules were phosphorylated—a remarkably high proportion. The species differences observed in relative amounts of histone were consistent in both stages of development. D. melanogaster differed from D. hydei and D. virilis in all histones except H2b, while the latter two species were generally similar. The interspecific variation in histone pattern was generally not correlated to differences in content of heterochromatin. The level of modification of H3 was, however, presumably an exception, as it was significantly lower for both larvae and adults in D. virilis than in the other two species. These differ from D. virilis in containing appreciably lower proportions of heterochromatic chromosome segments.     

Aurora, a non-mobile retrotransposon in Drosophila melanogaster heterochromatin

A novel retrotransposon, aurora, containing 324 by long terminal repeats (LTRs) was detected in Drosophila melanogaster as a 5 kb insertion in the heterochromatic Stellate gene. This insertion causes a 5 bp duplication of the integration site. Southern analysis and in situ hybridization data show that all detectable copies of aurora are immobilized in the D. melanogaster heterochromatin. However, mobile copies of aurora were revealed in the cuchromatin of D. simulans. The element was also found in various species of the melanogaster subgroup and in the D. virilis genome.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X70361 and X70362     

In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [^3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [³H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [³H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.     

A combined molecular and cytogenetic approach to genome evolution in Drosophila using large-fragment DNA cloning

Methods of genome analysis, including the cloning and manipulation of large fragments of DNA, have opened new strategies for uniting molecular evolutionary genetics with chromosome evolution. We have begun the development of a physical map of the genome of Drosophila virilis based on large DNA fragments cloned in bacteriophage P1. A library of 10,080 P1 clones with average insert sizes of 65.8 kb, containing approximately 3.7 copies of the haploid genome of D. virilis, has been constructed and characterized. Approximately 75% of the clones have inserts exceeding 50 kb, and approximately 25% have inserts exceeding 80 kb. A sample of 186 randomly selected clones was mapped by in situ hybridization with the salivary gland chromosomes. A method for identifying D. virilis clones containing homologs of D. melanogaster genes has also been developed using hybridization with specific probes obtained from D. melanogaster by means of the polymerase chain reaction. This method proved successful for nine of ten genes and resulted in the recovery of 14 clones. The hybridization patterns of a sample of P1 clones containing repetitive DNA were also determined. A significant fraction of these clones hybridizes to multiple euchromatic sites but not to the chromocenter, which is a pattern of hybridization that is very rare among clones derived from D. melanogaster. The materials and methods described will make it possible to carry out a direct study of molecular evolution at the level of chromosome structure and organization as well as at the level of individual genes.     

The hsp70 locus of Drosophila auraria (montium subgroup) is single and contains copies in a conserved arrangement

The restriction endonuclease pattern of a number of hsp70-homologous clones isolated from a library of heat shock cDNA from Drosophila auraria, a species belonging to the montium subgroup of the melanogaster species group, reveals two types of clones, A and B, differing in a single restriction site. Both types, as well as hsp70-specific probes derived from both hsp70 loci of Drosophila melanogaster, hybridize in situ with a single band at region 32 A of the 2L polytene arm, indicating a clustered organization of the hsp70 gene copies in D. auraria. The longest type B clone was sequenced and it was found that one strand contains an open reading frame (ORF) exhibiting great identity with a previously described hsp70 gene of D. auraria (now denoted as type A) and with its counterparts of D. melanogaster, while its second strand, unlike the type A clone, does not contain a long antiparallel coupled ORF (LAC ORF) because of a base substitution resulting in a premature stop codon. After additional data had been derived from isolation and characterization of hsp70-homologous genomic clones, together with Southern analysis of genomic DNA, we found that two hsp70 gene copies are present at the above locus of D. auraria with an inverted tandem repeat organization, while the presence of a third hsp70 gene is not clearly evident. The above results are compared with those observed at the homologous loci of some melanogaster subgroup species (D. melanogaster and its sibling species), in which, however, the hsp70 locus is duplicated, and with the more distantly related Dipteran Anopheles albimanus. Received: 22 May 1998; in revised form: 18 September 1998 / Accepted: 21 September 1998     

The pattern of polytene chromosome synapsis in Drosophila species and interspecific hybrids

The pairing of polytene chromosomes was investigated in Drosophila melanogaster, Drosophila simulans and their hybrids as well as in species of the D. virilis group and in F₁ hybrids between the species of this group. The study of frequency and extent of asynapsis revealed non-random distribution along chromosome arms both in interspecific hybrids and pure Drosophila species. It is suggested that definite chromosome regions exhibiting high pairing frequency serve as initiation sites of synapsis in salivary gland chromosomes.     

Aurora,a non-mobile retrotransposon in Drosophila melanogaster heterochromatin

A novel retrotransposon, aurora, containing 324 by long terminal repeats (LTRs) was detected in Drosophila melanogaster as a 5 kb insertion in the heterochromatic Stellate gene. This insertion causes a 5 bp duplication of the integration site. Southern analysis and in situ hybridization data show that all detectable copies of aurora are immobilized in the D. melanogaster heterochromatin. However, mobile copies of aurora were revealed in the cuchromatin of D. simulans. The element was also found in various species of the melanogaster subgroup and in the D. virilis genome.     

The Suppressor of fused gene, involved in Hedgehog signal transduction in Drosophila, is conserved in mammals

 The Suppressor of fused [Su(fu)] gene of Drosophila melanogaster encodes a protein containing a PEST sequence [sequence enriched in proline (P), glutamic acid (E), serine (S) and threonine (T)] which acts as an antagonist to the serine-threonine kinase Fused in Hedgehog (Hh) signal transduction during embryogenesis. The Su(fu) gene isolated from a distantly related Drosophila species, D. virilis, shows significantly high homology throughout its protein sequence with its D. melanogaster counterpart. We show that these two Drosophila homologs of Su(fu) are functionally interchangeable in enhancing the fused phenotype. We have also isolated mammalian homologs of Su(fu). The absence of the PEST sequence in the mammalian Su(fu) protein suggests a different regulation for this product between fly and vertebrates. Using the yeast two-hybrid method, we show that the murine Su(fu) protein can interact directly with the Fused and Cubitus interruptus proteins, known partners of Su(fu) in Drosophila. These data are discussed in the light of their evolutionary relationships. Received: 11 September 1998 / Accepted: 9 December 1998