Ionic interactions between proteins in nonequilibrium pH gradient electrophoresis: histones affect the migration of high mobility group nonhistone chromatin proteins

In two-dimensional gel electrophoresis of the high mobility group (HMG) proteins, it has proved necessary to use nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension rather than isoelectric focusing, because of the basic character of most of the HMG proteins [D. Tyrell, P. J. Isackson, and G. R. Reeck (1982) Anal. Biochem. 119, 433-439]. In this paper it is reported that in samples that contain histones, the mobilities of HMG proteins (particularly HMG-1, HMG-2, and HMG-E) are severely distorted in NEPHGE. This presumably results from formation of complexes between histones and HMG proteins through ionic interactions. Analysis of HMG proteins by NEPHGE/sodium dodecyl sulfate-gel electrophoresis is thus precluded in samples containing histones. Our results raise the possibility of similar artifacts occurring in NEPHGE (or isoelectric focusing) analysis of other proteins with regions of high charge density.     

Chromosomal replication in Drosophila virilis

DNA fiber autoradiography was used to determine parameters underlying the DNA replication of the eukaryotic chromosome in Drosophila diploid brain cells in organ culture. The average rate of fork movement, estimated from 4 different labelling intervals, is 0.35 μm/min at 25 ° C. Of the tandem arrays 93% show patterns which are compatible with bidirectional replication, 7% show unidirectional replication. The unidirectional mode of replication is interpreted as being a consequence of the experimental schedule (using hot-cold pulse labelling) combined with the occurrence of termination signals. — Some autoradiograms showed the expected two grain tracks of different densities; others showed only a high density track. The latter were most prominent in arrays of short replicons (<10 μm) which correlate with replicating satellite sequences. — The majority of replicons fall into size classes < 100 μm. The frequency distribution is skewed towards larger replicon sizes; it spans 2–238 μm, has a mean of ˉx = 35.6 μm and a median of = 21.0 μm. If the distribution is corrected for supposed satellite replicons, the median increases to = 31.0 μm. — In experiments using warmhot pulse labelling, arrays were scored which must have been a consequence of fixed termination signals. Furthermore, grain tracks diverging from weak labelled centers often have different lengths, indicating that these replicons contain two diverging replicating sections of unequal length. Presented to Professor Dr. Wolfgang Beermann on the occasion of his 60th birthday with my best wishes     

Chromosomal replication in Drosophila virilis

About half of the diploid genome of D. virilis is -heterochromatic (Heitz, 1934) and contains the satellite sequences found in isopycnic CsCl density gradients (Gall et al, 1971; Steinemann, 1976). The thymidine incorporation behavior of this material in the course of S phase was monitored by autoradiography. Labelled interphase nuclei show three types of labelling patterns, label exclusively confined to either eu- or -heterochromatin, and simultaneous labelling of both fractions. Using the fraction of labelled mitotic index method, the duration of the DNA-synthetic period, t_s = 11.9 ± 4.3 h and G₂ period, t_{G2 + 1/2M} = 6.9 ± 3.8 h, were determined. On the assumption that the investigated brain cells belong to an exponentially growing cell population, the cell cycle is 22.9 h long and the G₁ period lasts t_G1=4.1 h. The a-heterochromatin begins to replicate later than euchromatin and continues alone after a phase of common replication of both fractions. Noteworthy is the asynchronous termination in the proximal -heterochromatic segments of different chromosomes. Within the S phase, the first 1 h of DNA replication is exclusively confined to euchromatin, followed by 8 h of replication in both eu- and -heterochromatin and terminated by 3 h of exclusive -heterochromatin replication. Thus euchromatin has a doubling time of about 9 h and -heterochromatin of about 11 h. The -heterochromatin of D. virilis is late and slow replicating.     

X chromosome DNA variation in Drosophila virilis.

Comparisons of polymorphism patterns between distantly related species are essential in order to determine their generality. However, most work on the genus Drosophila has been done only with species of the subgenus Sophophora. In the present work, we have sequenced one intron and surrounding coding sequences of 6 X-linked genes (chorion protein s36, elav, fused, runt, suppressor of sable and zeste) from 21 strains of wild-type Drosophila virilis (subgenus Drosophila). From these data, we have estimated the average level of DNA polymorphism, inferred the effective population size and population structure of this species, and compared the results with those obtained for other Drosophila species. There is no reduction in variation at two loci close to the centromeric heterochromatin, in contrast to Drosophila melanogaster.     

RNA synthesis in embryogenesis of Drosophila virilis]

The synthesis of different RNA fractions labelled by 3H-uridine has been studied at different developmental stages in Drosophila virilis by means of thermal phenol extraction and chromatography on oligo-alphaT-cellulose. There are two periods of intensive synthesis of poly-A containing (messenger) RNA in the course of embryogenesis. The former (6--12 hrs) is the period of gastrulation and stomodeum development and the latter (15--21 hrs) is the period of intensive differentiation of larval organs. The synthesis of poly-A free RNAs predominates at the stages of blastoderm (2--5 hrs) and mesoderm segmentation and embryo shortening (12--15 hrs).     

DNA sequence divergence in the Drosophila virilis group

DNA sequence divergence was analyzed in some sibling species of the Drosophila virilis group. Clones comprising about 0.1% of the genome DNA were selected at random from a D. virilis library for a comparative study on DNA from D. lummei, D. novamexicana, D. borealis, and D. lacicola. Blot hybridization experiments indicated that about 70% of DNA from D. lummei and D. novamexicana and less than 50% of DNA from D. borealis and D. lacicola share sequences that are homologous to DNA in D. virilis. This finding is in excellent agreement with the genealogical tree based on cytological studies (Throckmorton 1982). - Four plasmids with inserts which are present in one or a few copies per genome were hybridized in situ to polytene chromosomes. These experiments demonstrate that (1) homologous "unique" DNA sequences are localized exclusively in homologous bands and (2) homologous bands that appear to be identical in different species may contain different DNA sequences.     

Biogenic amines in Drosophila virilis under stress conditions

The effect of heat stress (38 degrees C) on the content of DL-beta-(3,4-dihydroxyphenyl)alanine (DOPA), dopamine, tyramine, octopamine, and their precursor Tyr was studied in adults of two lines of Drosophila virilis contrasting in their stress response. In individuals of line 101 responding to stress by a hormonal stress reaction, the contents of DOPA, dopamine, octopamine, and Tyr were lower than those of line 147 that did not respond to the stress. However, heat stress caused an increase in the contents of DOPA, dopamine, octopamine, and Tyr in line 101, whereas the equivalent titers in line 147 remain unchanged.     

The genetics of esterases in Drosophila. VIII. The gene regulating the activity of JH-esterase in D. virilis

The content of JH-esterase was assayed by radial immunodiffusion in Drosophila virilis pupae under normal conditions and under the effects of extreme factors. It was found that JH-esterase content is the same (not different from the control) in pupae showing a high activity of the enzyme and in those not showing it. These data are evidence for a gene controlling JH-esterase activity. It was also shown that a regulatory factor converts inactive into active JH-esterase when homogenates of pupae, with active and inactive forms, were mixed and incubated together. It was demonstrated that the source of the activating factor is the larval brain. Sublines 147-R and 147-I were produced by introducing the second chromosome pair of stocks 103 and 101, which are heat resistant, into the genome of individuals of stock 147, which is heat sensitive. Sublines 160-III, 160-IV, 160-V, and 160-VI were produced by introducing the third, fourth, fifth, and sixth chromosome pairs of stock 147 into the genome of stock 160S, which is heat-resistant. The results of analysis of JH-esterase activity and the viability of individuals of these sublines at high temperatures indicated that the gene regulating the activity of JH-esterase is located in the sixth chromosome of D. virilis.     

Drosophila virilis and its cosmopolitan relatives in urban islands

Populations of brewery Drosophila have been studied for 2 years. Species distributions diner between breweries, and are very different from those found in woodland and fruitmarkets. It is suggested that breweries form 'urban islands'. One of the commonest species found in breweries appears to be Drosophila virilis , which has not previously been found in Britain. Tests on various characteristics, including courtship song, have shown conclusively that this species is a strain of D. virilis.     

DNA-protein interactions in the Drosophila virilis mitochondrial chromosome.

The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila virilis was investigated by Me3 psoralen photoreaction of mitochondria isolated from embryos. After photoreaction the mtDNA was purified and the pattern of DNA cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The transcribed regions of the mtDNA molecule contained some uncross-linked regions, but such regions were infrequent and randomly distributed. In contrast, the A + T-rich region around the origin of replication of the mtDNA was usually protected from psoralen cross-linking. The data were best fit by two protected sites, each approximately 400 base pairs, compared to the four 400 base pair sites observed in the equivalent region of D. melanogaster mtDNA [Potter et al. (1980) Proc. Nat. Acad. Sci. USA 77, 4118-4122]. Thus this region of the mtDNA appears to be involved in a DNA-protein structure that is highly conserved even though the DNA sequence has diverged rapidly relative to protein-coding sequences.     

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     

piRNAs and epigenetic conversion in Drosophila

Transposable element (TE) activity is repressed in the Drosophila germline by Piwi-Interacting RNAs (piRNAs), a class of small non-coding RNAs. These piRNAs are produced by discrete genomic loci containing TE fragments. In a recent publication, we tested for the existence of a strict epigenetic induction of piRNA production capacity by a locus in the D. melanogaster genome. We used 2 lines carrying a transgenic 7-copy tandem cluster (P-lacZ-white) at the same genomic site. This cluster generates in both lines a local heterochromatic sector. One line (T-1) produces high levels of ovarian piRNAs homologous to the P-lacZ-white transgenes and shows a strong capacity to repress homologous sequences in trans, whereas the other line (BX2) is devoid of both of these capacities. The properties of these 2 lines are perfectly stable over generations. We have shown that the maternal transmission of a cytoplasm carrying piRNAs from the first line can confer to the inert transgenic locus of the second, a totally de novo capacity to produce high levels of piRNAs as well as the ability to induce homology-dependent silencing in trans. These new properties are stably inherited over generations (n > 50). Furthermore, the converted locus has itself become able to convert an inert transgenic locus via cytoplasmic maternal inheritance. This results in a stable epigenetic conversion process, which can be performed recurrently—a phenomenon termed paramutation and discovered in Maize 60 y ago. Paramutation in Drosophila corresponds to the first stable paramutation in animals and provides a model system to investigate the epigenetically induced emergence of a piRNA-producing locus, a crucial step in epigenome shaping. In this Extra View, we discuss some additional functional aspects and the possible molecular mechanism of this piRNA-linked paramutation.     

Searching for a Common Centromeric Structural Motif: Drosophila Centromeric Satellite DNAs Show Propensity to form Telomeric-Like Unusual DNA Structures

The molecular basis of centromere formation in a particular chromosomal region is not yet understood. In higher eukaryotes, no specific DNA sequence is required for the assembly of the kinetochore, but similar centromeric chromatins are formed on different centromere DNA sequences. Although epigenesis has been proposed as the main mechanism for centromere specification, DNA recognition must also play a role. Through the analysis of Drosophilacentromeric DNA sequences, we found that dodeca satellite and 18HT satellite are able to form unusual DNA structures similar to those formed by telomeric sequences. These findings suggest the existence of a common centromeric structural DNA motif which we feel merits further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.