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We have established improved conditions for separating the chromosomes of Plasmodium falciparum by pulsed field gradient gel electrophoresis (PFG) using a contour-clamped homogenous electric field (CHEF) apparatus. Thirteen clearly separable chromosomal bands were reproducibly isolated from the strain FCR3 and their sizes have been determined. Evidence that indicates one band may contain two chromosomes is presented. The relationship between the PFG separable DNA and the number of unique chromosomes in P. falciparum is considered. We have established a relationship between the maximum resolvable sizes of the chromosomes and the pulse times. The chromosomal location of twenty-seven P. falciparum DNA probes is also reported.

The distribution of Z-form DNA along the length of metaphase chromosomes of Indian muntjac was studied by indirect immunofluorescence procedures using an antibody specific to the Z-DNA conformation. Several fixation conditions were compared for reproducible detection of Z-DNA in isolated metaphase chromosomes. Fixation of chromosomes with 45% acetic acid alone gave reproducible reactivity with the antibody. When fixation was done either with Carnoy's solution (3:1 methanol:acetic acid) or with 75% alcohol alone, the antibody binding was at background level. Acetic acid-fixed chromosomes exhibited intense fluorescence both at C-band heterochromatin and at nucleolus organizer regions (NORs). The euchromatic regions had weakly, but clearly, stained bands, which were quite similar to the chromomycin A3 R-bands. After treatment with topoisomerase I, the immunofluorescence at NORs and R-bands disappeared, but only a slight decrease in immunofluorescence intensity was observed at C-band regions. We suggest that this difference in the immunoreactivity of NORs and R-bands from C-bands reflects a difference in gene activity among these regions. Possible molecular mechanisms involved in Z-DNA immunoreactivity are discussed, based on SDS-polyacrylamide gel electrophoretic analysis of chromosomal proteins after extraction of metaphase chromosomes with different fixative solutions.

The purpose of this study is to determine the kinetics of the replication of intrachromosomal versus extrachromosomal amplified dihydrofolate reductase (DHFR) genes. Previous studies reported that the DHFR gene, when carried intrachromosomally on a homogeneously staining region, replicates (as a unit) within the first 2 h of the S phase of the cell cycle. We wished to determine if the extrachromosomal location of the amplified genes carried on double minute chromosomes effects the timing of their replication. Equilibrium cesium chloride ultracentrifugation was used to separate newly replicated (BUdR-labeled) DNA from bulk DNA in a synchronized cell population. Hybridization with the cDNA for the DHFR gene allowed us to determine the period of time within the cell cycle in which the DHFR DNA sequences were replicated. We found that, in contrast to intrachromosomal dihydrofolate reductase genes that uniformly replicate as a unit at the beginning of the S phase of the cell cycle, dihydrofolate reductase genes carried on double minute chromosomes (DMs) replicate throughout the S phase of the cell cycle. These results suggest that control of replication of extrachromosomal DNA sequences may differ from intrachromosomal sequences.

To study whether specific DNA sequences are associated with nuclear membranes, residual DNA was extracted from DNase-treated nuclear envelopes prepared from erythrocytes of adult chickens (Gallus domesticus). This DNA was then blunt-end ligated into a bacterial plasmid vector. DNA blot analysis and nucleotide sequence determination revealed that approximately 30% of the cloned fragments consisted of different multiples of a 41-42 bp tandemly repeated, partially symmetrical sequence. In situ hybridization to chicken chromosomes demonstrated that the sequence was located primarily on microchromosomes, although some hybridization was also observed to macrochromosomes 7 and 8. Digestion of chicken DNA with any of a number of restriction enzymes did not completely reduce the intensity of a high molecular weight band to which the repeated sequence hybridized. These results, along with those obtained from in situ hybridization, suggested that many copies of this sequence are organized into large tandem arrays, and are not dispersed in many shorter repetitive blocks throughout the chicken genome. Although the repetitive sequence constituted approximately 10% of the chicken genome, it did not hybridize to quail or turkey DNA.

A simple and rapid procedure to isolate clones carrying sequences from a specific region of the polytene chromosome of Drosophila is demonstrated. The procedure involves microdissection of the region of interest, amplification of the DNA by PCR using a primer designed to prime the synthesis nonspecifically, labeling of the amplified DNA using the random primer method, and screening of a standard library with the probe to identify and isolate clones carrying sequences homologous to the dissected region. This procedure has the potential to replace the difficult procedure of microcloning, as well as facilitate chromosome walking.

The expression throughout development of a long DNA segment from the Drosophila genome was investigated. A near continuum of 800 kb DNA was cloned by extending a 90 kb previous chromosome walk, spanning the rudimentary locus (r), and by initiating 2 chromosome walks from an isolated DNA clone located next to the r locus. Hexanucleotide-primed single strand cDNAs were synthesized from poly(A)+ RNAs prepared from staged embryos, larvae, pupae, males, females, heads from adults and thoraces plus abdomens from adults. The representativity of the cDNAs with regard to the whole length and the molar abundance of a selected gene mRNA was checked. These cDNAs were then used to probe Southern transfers of the cloned DNA after digestion with combinations of restriction enzymes. The limits of the expressed parts of the cloned DNA and their developmental profiles were then deduced. Data show that, in contrast to what was known on the basis of genetic analysis, this region of the Drosophila genome is densely transcribed. Numerous parts of the cloned DNA are found to be stage-, or sex- or territory-specifically expressed. Data are consistent with a view of the organization of this representative part of the Drosophila genome according to which the expressed regions tend to be clustered, whereas no tendency for transcription units displaying comparable specific spatial, temporal or sex expression patterns to be arranged in groups is noted.

We have established a method using a conventional cell sorter equipped with a single argon laser to sort intact human chromosomes that can be used as a source for the production of giant DNA fragments. Various improvements were made to both the equipment and sorting method to enhance the sorting resolution and avoid destruction of chromosomal DNA. Using this improved method chromosomes 21 and 22 were sorted from the B-lymphoblastoid line GM00130B, digested with the rare cutting restriction endonuclease NotI, and analyzed by pulsed field gel electrophoresis followed by Southern hybridization using the Alu repetitive sequence as a probe. More than 25 discrete NotI giant DNA fragments ranging from 50 kb to longer than 2.5 Mb were separated and the size distribution pattern was unique for each chromosome, indicating successful sorting of intact chromosomes. The cumulative size of these Alu-positive NotI DNA fragments were 22.7 Mb and 25.5 Mb for chromosomes 21 and 22, respectively. These values are 47% and 49% of the estimated size of chromosomes 21 (48 Mb) and 22 (52 Mb).

Sedimentation profiles for chromosomal DNA from unirradiated and X-irradiated yeast cells of wild type and rad 52 strains are presented. These profiles indicate that, whereas wild type strains rejoin DNA double-strand breaks, rad 52 strains apparently do not. These data suggest that the rad 52 mutant lacks a repair system for X-ray induced damage and are consistence with the proposal that an unrepaired chromosome break leads to reproductive cell death.

Spectrofluorometric characteristics of DNA-RO4 complexes have been studied in order to evaluate the possibility of employment of RO4 (a bis-intercalating dye) for the determination of the DNA base composition. The results have shown the usefulness of this compound for DNA with AT percentage between 30% and 60%. RO4 has been also applied to the study of the chromosome banding mechanism.

Molecular and cytogenetical analyses of three sibling species of Australian grasshopper, Atractomorpha australis, A. species-1 and A. similis, resolves one of the long standing problems of highly repeated DNA. In this system satellite DNA functions in regulating the level and position of recombination, irrespective of whether the repeated DNA is located in telomeric or centric regions. Even though the three species do not differ in their euchromatic genome sizes, their relative DNA contents are 1.00/1.10/1.41, the difference in genome size being due solely to visible centric or telomeric blocks of heterochromatin. Antibiotic analytical and preparative ultracentrifugation, in situ hybridization and renaturation kinetic analyses reveal that a large cryptic satellite of A. similis constitutes the heterochromatic telomeric blocks of nearly all autosomes and that the DNA of this satellite is highly repeated. Comparison of these grasshopper data with published literature of heterochromatic rearrangements in Drosophila and with heterochromatin distribution and recombination patterns in diploid plant species reveals that in every case heterochromatin is implicated in some form of alteration in the meiotic recombination system.

An abundant tandem repeat has been cloned from genomic DNA of the merlin (Falco columbarius). The cloned sequence is 174 bp in length, and maps by in situ hybridization to the centromeric regions of several of the large chromosomes within the merlin karyotype. Complementary sequences have been identified within a variety of falcon species; these sequences are either absent or in very low copy number in the family Accipitridae. The cloned merlin repeat reveals highly polymorphic restriction patterns in the peregrine falcon (Falco peregrinus). These polymorphisms, which have been shown to be stably inherited within a family of captive peregrines, can be used to differentiate the Greenland and Argentina populations of this endangered raptor species.

Metaphase chromosomes from Chinese hamster V-79 cells have been stained with propidium iodide and their DNA content was measured by flow cytometry. The chromosomes in suspension must be stabilized during the preparation to preserve their morphological integrity. In order to obtain high resolution in the flow karyotype, several modifications were made to the method used to prepare chromosome suspensions. The influence of several ions, detergents and detergent mixtures was investigated. Magnesium (3-5 mM) together with sodium (10 mM), Tris (10 mM) and Triton X-100 (0.4%) gave the best results. In contrast, sodium and potassium exerted no effect, while calcium had a negative effect on the preparation as measured with flow cytometry. Compared with a solution of potassium chloride + propidium iodide, chromosomes prepared in Tris buffer resolved better with less background. A histogram quality factor which contains the variables peak height, depth of valley and coefficient of variation, was used to quantitate the histograms.

Clusters of the tetranucleotide GATA are found throughout the mouse genome with a major concentration on the Y chromosome. In Drosophila melanogaster, by contrast, they have a significant concentration on the X chromosome. Largely on the basis of these sex chromosomal concentrations and on their transcriptional activity in the mouse, these simple sequence tracts have been thought to be important in sex-determining and X inactivation mechanisms in both vertebrates and invertebrates. In every tested case the interpretations of the data have been difficult and sometimes the data themselves have been conflicting. We demonstrate in this paper that significant tracts of (GATA)n are totally absent from ovine and bovine genomes and point out that none of the major clusters of these repetitive sequences are near any of the sex-determining genes in D. melanogaster. We conclude therefore that (GATA)n sequences are not conserved over long evolutionary time periods as has previously been thought. Their absence from at least two mammalian genomes places severe constraints on their possible functions.

Antisera and antibodies against Z-DNA were used on metaphasic fixed chromosomes of a mammal, Gerbillus nigeriae (Gerbillidae, Rodentia). By indirect immunofluorescence and indirect immunoperoxidase labelling a heavy staining was detected in a fraction of the R-band positive heterochromatic segments, which are presumed to be rich in G-C base pairs. A weak and non homogeneous staining was also observed on euchromatic segments. The presence of a left-handed Z-DNA in mammalian chromosomes is discussed.

Cytochemical quantitative methods were used to investigate DNA protein contents of mouse metaphase plates during an alkaline C-banding procedure ( Sumner et al., 1971). Cytochemical stains and reactions for DNA and for total protein content were used to quantitatively assess the sequential involvement (losses) of DNA and protein during the appearance of the classic C-banding pattern which was monitored with Giemsa staining. The data point the preferential loss of DNA from euchromatic regions of chromosomes as the main cause of the C-banding pattern appearance. The effect of chromosomal protein is more likely indirect and perhaps tied to some specific interaction with centromeric DNA that contributes to DNA retention in C-bands. Following the C-banding procedure it was possible to differentially stain the centromeric area with Feulgen and GCA and even with non-fully specific stain for DNA such as methylene blue.

Nuclear DNA contents were estimated by microdensitometry in five species of Akodon rodents: Arodon molinae, A. dolores, A. mollis, A. azarae, Bolomys obscurus) and in three chromosomal varieties of A. molinae (2n = 42; 2n = 43, 2n = 22). The data obtained showed that the species with the highest DNA content was B. obscurus, followed in order of decreasing genome size by A. molinae, A. mollis, A. dolores and A. azarae. In A. molinae the forms with 2n = 42 chromosomes had the lowest and the forms with 2n = 44 the highest amount of DNA, while the forms with 2n = 43 had intermediate DNA contents. The variation in DNA amount detected in A. molinae was interpreted as a phenomenon of amplification occurring in the chromosomal areas involved in the chromosomal rearrangement giving rise to the polymorphism exhibited by this species. The DNA contents of shared chromosomes (chromosomes with similar size, morphology and G banding pattern, which are found in two or more phylogenetically related species), were compared and correlated with values of total nuclear DNA. The information obtained indicates that: (a) shared chromosomes have variable amounts of DNA: (b) in a given species there is a correlation between the amount of nuclear and chromosomal DNA in most shared chromosomes (and perhaps in most of the chromosomal complement), e.g., the higher the amount of nuclear DNA, the higher the content of DNA in shared chromosomes; (c) some chromosomes may undergo processes of amplification or deletion restricted to certain regions and usually related with mechanisms of chromosomal rearrangements.(ABSTRACT TRUNCATED AT 250 WORDS)

A method is described for excising by a glass knife single bands of isolated polytene chromosomes of the salivary glands of Chironomus tentans larvae. DNA strands were isolated from cut-out bands and their contour lengths were determined on electron micrographs. The mean contour length of DNA strands isolated from the double band I-8A was about twice that of the single band I-11B, namely 63 versus 34 micrometers. The described method may be applicable for molecular studies on single bands (e.g., by DNA cloning).

Prior studies on subfractions of mouse and Kangaroo rat DNA have suggested that variations in base concentration within a given genome may not be great enough to account for Q-banding. To examine this with another species, calf DNA was subfractionated by CsCl ultracentrifugation into GC-rich satellites and the main band DNA was further fractionated into AT-rich, intermediate and GC-rich portions. The effect of varying concentrations of these DNAs on quinacrine and Hoechst 33258 fluorescence was examined. Although with both compounds there was less fluorescence in the presence of the GC-rich satellites than main band fractions, these results per se did not answer the question of whether the variation in base composition alone was adequate to account for chromosome banding. To answer this the fluorescence observed in the presence of DNA of a given base composition was related to the fluorescence observed in the presence of DNA of 40% GC content (F/F40). This allowed the derivation of a term B which indicated the relative change in fluorescence per 1% change in base composition of DNA. To determine the percent change in fluorescence observed in Q-banding, the photoelectric recordings of Caspersson et al. (1971) were used. From these data we conclude: 1. Quinacrine is twice as sensitive to changes in base composition as Hoechst 33258. 2. Variation in the base content of DNA along the base content of DNA along the chromosome is sufficient to account for most Q-banding, except possibly for some of the extremes of quinacrine fluorescence. This was further examined with daunomycin. Even though daunomycin gives good fluorescent banding, DNAs varying in base composition from 100 to 40% GC content all resulted in the same relative fluorescence of 0.03. However, in the presence of poly (dA-dT) the relative fluorescence was 0.85, indicating a great sensitivity to very AT-rich DNA. This suggests that with daunomycin and possibly other fluorochromes, stretches of very AT-rich DNA may be more important in fluorescent banding than simple variation in mean base composition.

A new approach is presented for the indirect hybridocytochemical localization of specific nucleic acid sequences in microscopic preparations. The method is based on the application of probes modified with N-acetoxy-2-acetylaminofluorene. After hybridization, the 2-acetylaminofluorene-labelled probes are recognized by antibodies directed against modified guanosine and visualized immunocytochemically. This procedure has been optimized on two model objects: mouse satellite DNA in interphase nuclei and chromosomes, and kinetoplast DNA in Crithidia fasciculata. A first application that may be of clinical importance is given by the detection of human cytomegalovirus in infected human lung fibroblasts. Other potentials of this procedure are discussed. Its advantages are: (1) the simple, rapid and reproducible labelling procedure; (2) the high stability of both label and modified probes; (3) the feasibility of labelling both double-stranded (ds) and single-stranded (ss) probes (DNA as well as RNA); (4) the rapid and sensitive detection of hybrids.

A small proportion (0.1-0.5%) of the total DNA content of native Chinese hamster metaphase chromosomes is protected from nucleolytic degradation following the removal of histones by extraction with either 0.2 N HCl or 2 M NaCl, and remains attached to the nonhistone protein core. Acid extraction followed by DNase I digestion leads to small fragments of 10-30 bases. Salt extraction followed by micrococcal nuclease digestion gives approx. 140 b.p. fragments which are undistinguishable in size from nucleosome core DNA fragments. Furthermore, DNase I treatment of salt extracted chromosomes gives DNA fragments containing single strands which are multiples of 10 bases in length, again characteristic of the nucleosome structure. Reassociation kinetics using the 32P-labelled 140 b.p. fragments as probes suggests they are enriched for rapidly reassociating sequences.

Quantitative measurement of DNA migration in gel electrophoresis requires precisely controlled homogeneous electric fields. A new electrophoresis system has allowed us to explore several parameters governing DNA migration during homogeneous field pulsed field gel (PFG) electrophoresis. Migration was measured at different switch times, temperatures, agarose concentrations, and voltage gradients. Conditions which increase DNA velocities permit separation over a wider size range, but reduce resolution. We have also varied the angle between the alternating electric fields. Reorientation angles between 105 degrees and 165 degrees give equivalent resolution, despite significant differences in DNA velocity. Separation of DNA fragments from 50 to greater than 7000 kilobases (Kb) can easily be optimized for speed and resolution based on conditions we describe.

Chromosomal DNA from 117 Trypanosoma congolense clones from 54 stocks, isolated from cattle introduced onto a ranch in Kilifi in the coastal area of Kenya, was fractionated by the orthogonal field alternation gel electrophoresis technique. The technique resolved chromosomes in the size range of 100 kb-1 Mb. The chromosome profile for cloned trypanosome populations was relatively stable with regard to number and size of the chromosome bands following transmission in mice, cattle, goats or tsetse flies. Only in one clone was a shift observed in the position of one medium-sized chromosome band following cyclical development in tsetse. On the basis of their chromosome profiles, the 117 clones could be divided into 18 distinct groups. Representative clones, randomly selected from 7 of the 18 chromosome profile groups were inoculated into steers and goats in order to raise variable antigen type (VAT) repertoire-specific infection sera. Cross-neutralization assays demonstrated that recovery sera from animals infected with a clone neutralized all the clones with an identical chromosome profile. This suggests that clones having an identical chromosome profile also express an identical VAT-repertoire (serodeme).

The 5-methylcytosine residues of L-cells have been labeled with [methyl-3H]-L-methionine and their chromatin localization studied using deoxyribonucleases. The kinetics of micrococcal nuclease digestion showed that the methylated cytosine residues are concentrated within regions resistant to nuclease digestion and preferentially missing from those regions between nucleosomes which are nuclease sensitive. Using DNA hybridization kinetic analysis, it is shown that 5-methylcytosine is abundant in highly repeated sequences but is also present in middle repetitive and unique sequence DNA.

Computer programs for high resolution slit-scan chromosome analysis in flow cytometry are introduced. A modular program library, SCAN, contains programs for single and dual parameter data acquisition, correction of recorded profiles and computation of histograms of various parameters. Using a minicomputer system, data acquisition is programmed in assembler to realize high input rates and real-time histogram calculation. Software for the processing of recorded profiles has been written in FORTRAN and allows extensions or alterations for different objectives. A sample run recording bicolor fluorescence profiles from metaphase chromosomes demonstrates the main features of the software.

Native rat liver NHC protein-DNA interactions have been investigated by use of a nitrocellulose filter assay sensitive in detection of protein-DNA complexes. Optimal conditions for DNA-protein interactions occurs at low ionic strength conditions (110 mM phosphate buffer). A fraction of NHC proteins was enriched 25-fold by their affinity for rat DNA immobilized on cellulose columns under these conditions. At higher ionic strength (260 mM-0.04M phosphate buffer and 0.15 M sodium chloride), this fraction binds approximately sevenfold less to rat DNA but with a substantial increase in stability of the complexes. Equilibrium competition experiments indicate that at the higher ionic strength there is a considerable DNA sequence specificity of the rat DNA binding NHC protein. Since rat DNA contains three components as defined by their reassociation kinetics: single copy DNA (C0t1/2pure = 1.6 times 103); middle repetitive DNA (C0t1?1PURE = 1.1); and highly repetitive (C0t1/2pure smaller than 0.02). The two former were isolated and employed in the DNA binding assays. At the high ionic strength criterion, the rat DNA binding NHC proteins showed a substantial preference for a subset of middle repetitive DNA sequences. This suggests a preferential interaction between a class of NHC proteins and a class of middle repetitive DNA sequences.

With the aid of Q- and N-banding techniques we investigated the relationship between the length of satellite stalks, the appearance of N-bands and the frequency of satellite association of individual acrocentric chromosomes in the cells of seven individuals, including one male with a satellited and small Y-chromosomes. The appearance of N-bands, seemed to be a constant and characteristic property of individual acrocentric chromosomes, independent of the status of concentration of the chromosomes at metaphase. The homolog with longer satellite stalks had larger N-bands and participated in satellite association at a higher frequency than the one with shorter stalks. It appeared that N-bands were present along the whole length of the satellite stalk, the size of which could possibly reflect the amount of rDNA present in the nucleolar organizers in human chromosomes.

pDm plasmids were constructed from D. melanogaster and pSC101 DNAs by a modification of the EcoR1-ligase method which insured that each hybrid molecule contained a single segment of D. melanogaster chromosomal DNA (Dm segment). The sequences in the Dm segments of six cloned pDm DNAs were mapped within the D. melanogaster polytene chromosomes by in situ hybridization, and their repetition frequencies within the Dm segment and within the genome were determined. Four of these segments consist of sequences that are confined to single chromomeric regions in the polytene chromosomes and exhibit little or no repetition. The characteristics of this group, and also two of three Dm segments analyzed earlier (Wensink et al., 1974), are inconsistent with tandem repetition models of the chromomere. By contrast, the other two Dm segments contain moderately repetitive sequences that are located in the heterochromatin. One of these appears to be a segment of the Y chromosome in which about half the sequences are nonrepetitive and half are repeated about 33 times per genome, though they are not repeated within the segment. The second contains the DNA coding for 18 and 28S rRNA.

Mitotic chromosomes of L cells (metaphase plates) were dehistonized by centrifugation through a layer of 2 M NaCl and then treated with restriction endonuclease Bam HI. Alternatively, they were pretreated with EcoRI endonuclease, dehistonized, and additionally digested with EcoRI or HindIII. The DNA remaining attached to the axial structure of the chromosomes was isolated and investigated in renaturation experiments. It was found to be enriched in reiterated base sequences belonging to the satellite and to abundant intermediate repeats. The CsCl density gradient ultracentrifugation of this DNA separated the satellite from the fraction containing intermediate repeats.

Scanning cytophotometric measurements on 3200 telophase and 1700 interphase nuclei (Feulgen-stained) in vegetative and reproductive buds of Rhoeo discolor revealed a transitory increase in staining intensity in more than half of the cells in early differentiating floral buds. The differences between the vegetative and floral nuclei are significant at the 0.001 level of probability and highly reproducible, independent of the type of hydrolysis used. We suggest that the different Feulgen extinction values reflect different nuclear DNA amounts, because methodical errors can fairly be excluded. The occurrence of an extra DNA (control DNA) of the kind of the floral DNA' detected by Wardell and Skoog (1973) and Wardell (1976) in tobacco is discussed.

The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobility-group protein 1 with DNA has also been studied. 1. Circular dichroism results indicate that in the presence of salt both proteins are 40-50% helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4. 2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding. 3. Nuclear magnetic resonance spectra of compelxes between high mobility group protein 1 and DNA demonstrate that a low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA.

The intact interphase genome of Drosophila melanogaster has been isolated by sucrose gradient centrifugation after gentle lysis of tissue culture cells in 0.9 M NaCl-0.4% nonidet P40. The non-viscous folded DNA sediments as a single broad 5000S peak in a complex with RNA (a fraction of the nuclear nascent RNA) and protein (all of the four intranuclesome histones: H2A, H2B, H3, and H4). The folded DNA is supercoiled and can be relaxed to slower sedimenting forms either by intercalating ethidium or by nicking with DNAase I. Incomplete DNAase treatment gives partially relaxed complexes, indicating that each nick relaxes only a stretch of DNA (defined as a supercoiled DNA loop) without affecting the superhelical content of the rest of the genome. The concentration of superhelices in the Drosophila folded DNA is the same as in the E. coli and SV40 closed circular DNAs-that is, about one negative turn every 200 base pairs (bp) in 0.15 M NaCl at 26 degrees C. The estimated average size of the supercoiled DNA loops, about 85,000 bp, equals the size of the larger Drosophila chromomeres. Ethidium intercalation in 0.9 M NaCl both removes the negative superhelical turns and dissociates the four histones from the DNA. The four histones are dissociated in equimolar concentrations, and the relative proportion of histones displaced from the DNA is a function of ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration which removes all of the negative superhelices. Thus the data strongly suggest that the rotation of the Watson Crick helix which accompanies ethidium intercalation causes the loss of nucleosomes from the DNA. The results are interpreted in terms of a model for the folded Drosophila genome which has the DNA constrained (by both protein-DNA and RNA- DNA interactions) into independent supercoiled loops containing on the average 400 nucleosomes per loop. Each nucleosome is composed of a histone core with the DNA wound around it in a 360 degrees left-handed toroidal supercoil; each nucleosome toroidal supercoil plus its relaxed internucleosome DNA contains, on the average, 200 bp.

Balbiani rings in Chironomus are large puffs on salivary gland polytene chromosomes that contain functionally related, but nonidentical genes that code for tissue-specific secretory polypeptides. In situ hybridization was used to select a recombinant plasmid (pCtBR1-1) that contained an insert of Chironomus tentans genomic DNA that originated from Balbiani ring 1. Mapping with restriction endonucleases demonstrated that the insert was 385 bp (base pair) and it contained duplicate clusters of certain cleavage sites about 250 bp apart. This repeat was shown to be part of tandem sequence arrays in the genome by hybridization of radioactive pCtBR1-1 to nitrocellulose blots containing limit and partial restriction endonuclease digests of nuclear DNA. Subsequent sequence analysis of the cloned DNA confirmed the presence of one complete copy of a 246-bp repeat comprised of a 114-bp internally nonrepeating segment and a 132-bp segment containing four 33-bp subrepeats. The subrepeats apparently evolved from a simple 9-bp sequence encoding a consensus tripeptide (Lys-Pro-Ser) in which the first two codons (AAA-CCA) were highly conserved at the nucleotide level. Comparisons between intragenic and interspecific (BRb in Chironomus thummi) copies of corresponding sequences revealed that, during the evolution of these tandemly repeated protein-coding sequences, internally nonrepeated segments were highly conserved and most likely became interspersed by variable segments containing subrepeats that arose from reduplication and divergence of 9-bp repeats.

A 12 kb fragment of Drosophila melanogaster DNA cloned in a lambda phage, lambda T-A, is shown by in situ hybridization to contain sequences homologous to DNA at the extreme ends of each of the polytene chromosomes and to the pericentric sequences present in the beta heterochromatin. This pattern of hybridization is seen for each of the four D. melanogaster stocks that have been studied. Most of the sequence in lambda T-A shows no detectable homology to DNA within the banded chromosome arms. (The only exception is what appears to be a short mobile element inserted in lambda T-A. This portion of lambda T-A hybridizes with internal arm sites that vary from stock to stock). Analysis of restriction fragments of genomic DNA indicates that the sequences in lambda T-A are homologous to complex sets of repeated sequences that differ from stock to stock in D. melanogaster. Some, but not all, of the members of these sets are underreplicated during polytenization in D. melanogaster. The sequences in lambda T-A are also homologous to complex sets of repeated sequences in the genomes of other Drosophila species belonging to the melanogaster species group. Pericentric and telomeric localization is conserved in these related species, although analysis of DNA fragments shows marked changes in sequence organization on a finer scale. The constancy of the localization of lambda T-A-homologous sequences to telomeric and pericentric regions suggests that these sequences serve a function in those regions.