Drosophila melanogaster H1 histone is phosphorylated stably.

Phosphorylation of histone H1 is developmentally regulated in Drosophila spp. It cannot be detected in preblastoderm embryos or polytene salivary gland cells, but in cellular blastoderm, postblastoderm embryo, and amitotic adult head nuclei, it occurs with a frequency of roughly 4 x 10(5) molecules per nucleus. We used pulse-labeling to study the relationship between H1 synthesis and modification in cultured cells. These results reveal that the H1-associated phosphate is stable and suggest that Drosophila H1 is synthesized, translocated to the nucleus, associated with chromatin, and then phosphorylated. Partial tryptic digestion of Drosophila H1 revealed that the phosphorylation site is located within the globular, central domain of the protein. Thus, the developmentally regulated phosphorylation of Drosophila H1 presents two contrasts with previously studied H1 phosphorylation. It is not correlated with DNA replication, and it is located in the central domain of the protein.     

Sequence arrangement of the rDNA of Drosophila melanogaster.

The sequence arrangement of genes coding for stable rRNA species and of the interspersed spacers on long single strands of rDNA purified from total chromosomal DNA of Drosophila melanogaster has been determined by a study of the structure of rRNA:DNA hybrids which were mounted for electron microscope observation by the gene 32-ethidium bromide technique. One repeat unit contains the following sequences in the order given. First, an 18 S gene of length 2.13 +/- 0.17 kb. Second, an internal transcribed spacer (Spl) of length 1.58 +/- 0.15 kb. A short sequence coding for the 5.8S and perhaps the 2S rRNA species is located within this spacer. Third, the 28S gene with a length of 4.36 +/- 0.23 kb. About 55% of the 28S genes are unbroken or continuous (C genes). However, about 45% of the 28S genes contain an insertion of an additional segment of DNA that is not complementary to rRNA (l genes). The insertion occurs at a reproducible point 2.99 +/- 0.26 kb from the junction with Spl. The insertions are heterogeneous in length and occur in three broad size classes: 1.42 +/- 0.47, 3.97 +/- 0.55, and 6.59 +/- 0.62 kb. Fourth, an external spacer between the 28S gene and the next 18S gene which is presumably mainly nontranscribed and which has a heterogeneous length distribution with a mean length and standard deviation of 5.67 +/- 1.92 kb. Short inverted repeat stems (100-400 nucleotide pairs) occur at the base of the insertion. It is known from other studies that I genes occur only on the X chromosome. The present study shows that the I and C genes on the X chromosomes are approximately randomly assorted. The sequence arrangement on the plasmid pDm103 containing one repeat of rDNA (Glover et al., 1975) has been determined by similar methods. The I gene on this plasmid contains an inverted repeat stem. The occurrence of inverted repeat sequences flanking the insertion supports the speculation that these sequences are translocatable elements similar to procaryotic translocons.     

Phylogenetic relationships of Drosophila melanogaster species group deduced from spacer regions of histone gene H2A-H2B

Nucleotide sequences of the spacer region of the histone gene H2A-H2B from 36 species of Drosophila melanogaster species group were determined. The phylogenetic trees were reconstructed with maximum parsimony, maximum likelihood, and Bayesian methods by using Drosophila pseudoobscura as the out group. Our results show that the melanogaster species group clustered in three main lineages: (1). montium subgroup; (2). ananassae subgroup; and (3). the seven oriental subgroups, among which the montium subgroup diverged first. In the third main lineage, suzukii and takahashii subgroups formed a clade, while eugracilis, melanogaster, elegans, ficusphila, and rhopaloa subgroups formed another clade. The bootstrap values at subgroup levels are high. The phylogenetic relationships of these species subgroups derived from our data are very different from those based on some other DNA data and morphology data.     

Chromatin fine structure of the histone gene complex of Drosophila melanogaster.

We have used salt extractions of nuclei and long agarose gels to dissect the chromatin fine structure of the histone gene repeat of Drosophila melanogaster. Extraction of nuclei with 0.35 M KCl removes many non-histone chromosomal proteins but does not significantly disturb the overall nucleosome arrangement of the repeat unit. After extraction of nuclei with 0.55 M KCl, which also removes histone Hl, the basic arrangement of nucleosome core particles in the repeat unit is not greatly disturbed and the exposed DNA segments near the 5' ends of the histone genes are also retained. Extraction of nuclei with 0.75 M or higher KCl concentrations causes extensive nucleosome sliding and rearrangement with accompanying changes in the nucleoprotein organization of the histone gene complex and loss of the 5' hypersensitive sites. Our results indicate that the histone gene repeat displays a highly organized chromatin structure in vivo.     

Analysis of histone messenger RNA of Drosophila melanogaster by two-dimensional gel electrophoresis.

The kinetics of the hydrogen-deuterium exchange reactions of double-helical poly (rI) · poly (rC), single-stranded poly(rC) and poly(rI), inosine, and cytosine- 5′-phosphoric acid have been examined, at various temperatures in the range 20 °C to 52 °C, by stopped-flow ultraviolet spectrophotometry, in the region 270 to 300 nm. For the solution of double-helical poly(rI) · poly(rC), two first-order deuteration reactions were found: a fast one and a slow one. At 25 °C and at pH 7.0, the rate constant was 12.3 s^?1 for the fast reaction, and 0.13 s^?1 for the slow reaction. The rate constant of the fast reaction is nearly equal to that of the single-stranded poly(rC) (12.6 s^?1), and is assigned to the deuteration at the amino hydrogen (that is, free from the C · I hydrogen bond) of the cytosine residue. The slow reaction is attributable to the deuteration of the two hydrogens: the amino hydrogen of rC and imide hydrogen of rI, which are rapidly exchanging with each other within every rC · rI base-pair. From the observed temperature effect on this slow reaction rate, it has been concluded that there are two types of “opening process” that are relevant to the hydrogen exchange reaction; one of them is predominent in the range 47 °C to 52 °C and the other in the temperature region lower than 47 °C. The enthalpy (H) and entropy (S) differences of the “open” and “closed” forms in the former type process are ΔH = 167 kcal per mole and ΔS = 507 e.u., while in the latter ΔH = 8.1 kcal per mole and ΔS = 10 e.u..     

tRNA derived insertion element in histone gene repeating unit of Drosophila melanogaster.

Analysis of 41 histone homologous clones from an isogenic gene library of Drosophila melanogaster showed that non-histone fragments interrupt the histone repetitive clusters at several sites. Long (L) and short (S) forms of the repeating units are distinguished by the insertion of 240 bp into the spacer between H1 and H3 of the L units; Each form appears to be clustered with its own kind. The complete DNA sequence of the histone 5.0 kb repeating unit was determined. Five histone genes (H1, H2A, H2B, H3, H4) were identified in a repeating unit and several sequence blocks common to the five histone genes were found in the 5'- and 3'-regions. The insertion sequence of 240 bp was found to be similar to the Alu family, an element derived from tRNA.     

Developmental regulation of N-terminal H2B methylation in Drosophila melanogaster

Histone post-translational modifications play an important role in regulating chromatin structure and gene expression in vivo. Extensive studies investigated the post-translational modifications of the core histones H3 and H4 or the linker histone H1. Much less is known on the regulation of H2A and H2B modifications. Here, we show that a major modification of H2B in Drosophila melanogaster is the methylation of the N-terminal proline, which increases during fly development. Experiments performed in cultured cells revealed higher levels of H2B methylation when cells are dense, regardless of their cell cycle distribution. We identified dNTMT (CG1675) as the enzyme responsible for H2B methylation. We also found that the level of N-terminal methylation is regulated by dART8, an arginine methyltransferase that physically interacts with dNTMT and asymmetrically methylates H3R2. Our results demonstrate the existence of a complex containing two methyltransferases enzymes, which negatively influence each other's activity.     

Polymorphism and stability in the histone gene cluster of Drosophila melanogaster

Histone genes in Drosophila melanogaster are organized into repeats of 4.8 and 5.0 kb (Lifton et al., 1978). We find these repeat sizes in every one of the more than 20 Drosophila strains we have examined. Strains differ in the relative amounts of the two repeat types, with ratios varying from 11 to 14, the 5.0 kb repeat always present in equal or greater amounts than the 4.8 kb repeat. Restriction enzyme digestion and blotting analysis reveals that the strains also differ in a number of far less abundant fragments containing histone DNA sequences. In the Amherst and Samarkand strains, there are, in addition, many copies of 4.0 and 5.5 kb repeat-like fragments respectively. A series of stocks were made isogenic for single second chromosomes from the Amherst strain. The hybridization patterns of the histone DNA from these stocks containing different Amherst chromosomes are very similar but a number of differences in the minor fragments were seen. The stability of the histone locus restriction pattern was tested by following the DNA derived from a single second chromosome of the b Adhn² pr cn strain over a two year period. The restriction pattern of major and minor bands remained identical. Finally, histone loci distinguishable by their restriction pattern on blots were recombined with visible markers. These chromosomes will be useful in tracing the fate of specific histone loci during genetic manipulations.     

Molecular analysis of the ecdysterone-inducible 2B5 "early'' puff in Drosophila melanogaster.

The 2B5 region of the X-chromosome in Drosophila melanogaster plays a developmentally important role in the ecdysterone-triggered response of the late third instar salivary gland. Using a combination of transposon-tagging and chromosomal walking techniques, we have isolated 231 kb of contiguous genomic DNA sequences corresponding to this region. We have more precisely aligned this DNA to the 2B1,2 to 2B5-6 interval of the cytogenetic map by locating the position of three well-characterized chromosomal breakpoints by in situ hybridization and genomic DNA blotting experiments. Labeled cDNA, synthesized from poly(A)+ RNA isolated from hormone-induced salivary gland and imaginal disc tissues and hybridized to the cloned DNA, demonstrated that the ecdysterone-inducible sequences mapped to DNA segments corresponding to the 2B3,4 to 2B5-6 interval. Although some of these sequences were inducible in only one tissue type, many were found to be inducible in both salivary glands and imaginal discs. RNA blotting experiments have detected a major 4.5-kb RNA which is hormone inducible in the larval salivary gland and whose quantitative induction is not inhibited by cycloheximide. Thus, the 4.5-kb RNA represents at least one product from the ecdysterone-responsive 2B5 "early' puff.     

Interaction of Drosophila melanogaster prohormone convertase 2 and 7B2. Insect cell-specific processing and secretion

The prohormone convertases (PCs) are an evolutionarily ancient group of proteases required for the maturation of neuropeptide and peptide hormone precursors. In Drosophila melanogaster, the homolog of prohormone convertase 2, dPC2 (amontillado), is required for normal hatching behavior, and immunoblotting data indicate that flies express 80- and 75-kDa forms of this protein. Because mouse PC2 (mPC2) requires 7B2, a helper protein for productive maturation, we searched the fly data base for the 7B2 signature motif PPNPCP and identified an expressed sequence tag clone encoding the entire open reading frame for this protein. dPC2 and d7B2 cDNAs were subcloned into expression vectors for transfection into HEK-293 cells; mPC2 and rat 7B2 were used as controls. Although active mPC2 was detected in medium in the presence of either d7B2 or r7B2, dPC2 showed no proteolytic activity upon coexpression of either d7B2 or r7B2. Labeling experiments showed that dPC2 was synthesized but not secreted from HEK-293 cells. However, when dPC2 and either d7B2 or r7B2 were coexpressed in Drosophila S2 cells, abundant immunoreactive dPC2 was secreted into the medium, coincident with the appearance of PC2 activity. Expression and secretion of dPC2 enzyme activity thus appears to require insect cell-specific posttranslational processing events. The significant differences in the cell biology of the insect and mammalian enzymes, with 7B2 absolutely required for secretion of dPC2 and zymogen conversion occurring intracellularly in the case of dPC2 but not mPC2, support the idea that the Drosophila enzyme has specific requirements for maturation and secretion that can be met only in insect cells.     

Perlecan domain V of Drosophila melanogaster. Sequence, recombinant analysis and tissue expression.

The C-terminal domain V of the basement membrane proteoglycan perlecan was previously shown to play a major role in extracellular matrix and cell interactions. A homologous sequence of 708 amino-acid residues from Drosophila has now been shown to be 33% identical to mouse perlecan domain V. It consists of three laminin G-type (LG) and epidermal growth factor-like (EG) modules but lacks the EG3 module and a link region found in mammalian perlecans. Recombinant production of Drosophila perlecan domain V in mammalian cells yielded a 100-kDa protein which was folded into a linear array of three globular LG domains. Unlike the mouse counterpart, domain V from Drosophila was not modified by glycosaminoglycans and endogenous proteolysis, due to the absence of the link region. It showed moderate affinities for heparin and sulfatides but did not bind to chick alpha-dystroglycan or to various mammalian basement membrane proteins. A single RGD sequence in LG3 of Drosophila domain V was also incapable of mediating cell adhesion. Production of a proteoglycan form of perlecan (approximately 450 kDa) in one Drosophila cell line could be demonstrated by immunoblotting with antibodies against Drosophila domain V. A strong expression was also found by in situ hybridization and immunohistology at various stages of embryonic development and expression was localized to several basement membrane zones. This indicates, as for mammalian species, a distinct role of perlecan during Drosophila development.