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By using improved pulsed field gel conditions, the karyotypes of several strains of the protozoan parasite Trypanosoma cruzi were analyzed and compared with those of Leishmania major and two other members of the genus Trypanosoma. There was no difference in chromosome migration patterns between different life cycle stages of the T. cruzi strains analyzed. However, the sizes and numbers of chromosomal bands varied considerably among T. cruzi strains. This karyotype variation among T. cruzi strains was analyzed further at the chromosomal level by using multicopy genes as probes in Southern hybridizations. The chromosomal location of the genes encoding alpha- and beta-tubulin, ubiquitin, rRNA, spliced leader RNA, and an 85-kilodalton protein remained stable during developmental conversion of the parasite. The sizes and numbers of chromosomes containing these sequences varied among the different strains analyzed, implying multiple rearrangements of these genes during evolution of the parasites. During continuous in vitro cultivation of T. cruzi Y, the chromosomal location of the spliced leader gene shifted spontaneously. The spliced leader gene encodes a 35-nucleotide RNA that is spliced in trans from a 105-nucleotide donor RNA onto all mRNAs in T. cruzi. The spliced leader sequences changed in their physical location in both the cloned and uncloned Y strains. Associated with the complex changes was an increase in the infectivity of the rearranged variant for tissue culture cells. Our results indicate that the spliced leader gene clusters in T. cruzi undergo high-frequency genomic rearrangements.

Many indirect methods have been developed to study the constitution and conformation of macromolecules inside the living cell. Direct analysis by Raman spectroscopy is an ideal complement to techniques using directly labelled fluorescent probes or of indirect labelling with mono- and polyclonal antibodies. The high information content of Raman spectra can characterize biological macromolecules both in solution and in crystals. The positions, intensities and linewidths of the Raman lines (corresponding to vibrational energy levels) in spectra of DNA-protein complexes yield information about the composition, secondary structure and interactions of these molecules, including the chemical microenvironment of molecular subgroups. The main drawback of the method is the low Raman scattering cross-section of biological macromolecules, which until now has prohibited studies at the level of the single cell with the exception of (salmon) sperm heads, in which the DNA is condensed to an exceptionally high degree. Ultraviolet-resonance Raman spectroscopy has been used to obtain single cell spectra (and F. Sureau and P. Y. Turpin, personal communication), but in this method absorption of laser light may impair the integrity of the sample. We have avoided this problem in developing a novel, highly sensitive confocal Raman microspectrometer for nonresonant Raman spectroscopy. Our instrument makes it possible to study single cells and chromosomes with a high spatial resolution (approximately less than 1 micron 3).

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.

When females of the newt Pleurodeles waltl, normally raised in our laboratory at 20 degrees C, were placed in 8 degrees C water for several days, striking modifications occurred in the structure of oocyte lampbrush chromosomes: numerous normal-type lateral loops were partially reduced in size and number, while hyperdeveloped loops of a new morphological type occurred at constant, reproducible loci. However, induction of these cold loops apparently was not accompanied by preferential RNA synthesis at their levels. Cold loops resulted from the decompaction of specific landmarks, the granular loops. Autoradiography revealed a significant drop in lampbrush chromosome transcriptional activity at 8 degrees C. Both structural and transcriptional modifications were fully reversible when oocytes were returned to normal temperature. These modifications are discussed in relation to processes which could determine the morphological appearance of landmark loops.

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.

We present, to our knowledge, the first extensively studied case of lymphoid L2 blast crisis of chronic myelogenous leukemia with a hand mirror cell (HMC) variant. Special stains revealed the leukemic cells to be terminal deoxynucleotidyl transferase positive by immunofluorescence and cytochemically positive for alpha-naphthyl acetate esterase and acid phosphatase (diffuse granular). Immunophenotyping identified the major leukemic cell population as B-cells that expressed CD10+, CD19+, and HLA-DR+. It was not possible to separate the HMC and the non-HMC leukemic population by gating various cell populations, dual staining, cytochemistry, or by terminal deoxynucleotidyl transferase. Gene rearrangements were observed in both Ig heavy-chain alleles and one T-cell antigen receptor gamma-subunit allele. The rearrangements occupied all of the cells, indicating that the HMC and non-HMC were of a common clonal origin. The patient had a mosaic karyotype, with 90% of the cells having t(9;22), t(8;14), and t(9;15) translocations, an additional chromosome 8, and deleted chromosomes 9 and 15. Antibodies to simian sarcoma-associated virus and baboon endogenous virus were isolated in the patient's peripheral blood plasma.

Investigating genes and their transcription products in nuclear compartments corresponding to one mammalian chromosome, the ribosomal genes 18S-28S and 5S were localized in PtK1 micronucleated cells and rRNA was characterized in sorted micronuclei containing single identified chromosomes. In situ hybridization revealed the presence of 18S-28S rRNA genes in two micronuclei per cell and 5S rRNA genes in four micronuclei per cell. Flow cytometry histograms of isolated micronuclei stained with Hoechst 33342 exhibited five peaks (a-e) in which peaks b and c, respectively, corresponded to chromosomes 4 and X. Restricted genomic DNA from sorted peak c micronuclei showed the presence of 28S gene sequences. Direct sorting of the micronuclei from each peak on nitrocellulose and their hybridization with the 18S-28S rDNA probe revealed that the rRNA genes were exclusively located in micronuclei containing X chromosomes. Northern blotting showed the presence of 18S-28S and 5S rRNAs in peak c micronuclei and their absence from peak b micronuclei. Consequently, these procedures allowed us to show the presence of ribosomal genes and the corresponding rRNA in micronuclei containing single X chromosomes, and the absence of rRNA from micronuclei that do not contain the ribosomal genes. In regards to the transcription of these genes, the micronuclei from peak c can be considered as functional interphase X chromosomes.

Twelve different human keratinocyte strains were transformed with recombinant plasmid pSV6-1 which contained an origin defective SV40 genome. When injected into athymic nude mice lines produced either squamous cell carcinomas (SCC) in all animals, SCC in some animals and epidermal cysts in others, or epidermal cysts only in all the animals. The tumourigenic capacity of the lines could be correlated with the chromosomal changes present initially in the transformed cells. Lines which produced SCC in all the animals within a short period of time all showed simultaneous loss of part of chromosomes 3p, 8p and 11p in one homologue. Lines which were not tumourigenic did not show these simultaneously appearing rearrangements. These specific rearrangements are acquired in vitro and the time taken for a recognisable tumour to appear is related to the proportion of such cells in the line. The rearrangement of the same chromosome regions in different tumourigenic cell lines suggests that genes in these regions are important in the development of squamous cell carcinoma, possibly by loss of heterozygosity, at particular loci.

The family of three structurally related autoantigens CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD) are the best characterized components of the human centromere, and they have been widely assumed to be components of the kinetochore. Kinetochore components are currently of great interest since this structure, which has long been known to be the site of microtubule attachment to the chromosome, is now believed to be a site of force production for anaphase chromosome movement. In the present study we have mapped the distribution of CENP-B in mitotic chromosomes by immunoelectron microscopy using two monospecific polyclonal antibodies together with a newly developed series of ultra-small 1-nm colloidal gold probes. We were surprised to find that greater than 95% of CENP-B is distributed throughout the centromeric heterochromatin beneath the kinetochore. This strongly supports other emerging evidence that CENP-B is specifically associated with alpha-satellite heterochromatin. Although in certain instances CENP-B can be seen to be concentrated immediately adjacent to the lower surface of the kinetochore, the outer plate remains virtually unlabeled. Similar analysis with a human autoimmune serum that recognizes all three CENP antigens reveals an additional unsuspected feature of kinetochore structure. In addition to recognizing antigens in the centromeric heterochromatin, the autoantiserum recognizes a concentration of antigens lateral to the kinetochore. This difference in staining pattern may reflect the presence of a "collar" of chromatin rich in CENP-C and/or CENP-A encircling the kinetochore plates.

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.

Human autoantibodies from a patient with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) were used to immunostain kinetochores on chromosomes in endosperm of the seed of the monocot Haemanthus katherinae Bak. Kinetochores of mitotic chromosomes and prekinetochores of interphase cells were specifically stained using conventional indirect immunofluorescence procedures as well as a nonfading immunogold-silver-enhanced technique and analyzed by fluorescence and video microscopy. In interphase, prekinetochores were either single or double structures depending on the stage of the cell cycle but became quadruple (two distinct stained dots on each chromatid) in mid-to-late prophase. In favorable preparations of prometaphase chromosomes, multiple subunits could be resolved within each sister kinetochore suggesting a compound organization. Western blot analysis demonstrated common epitopes in centromeric peptides of HeLa and Haemanthus cell extracts. Although the molecular mass of individual polypeptides differed in the two species, the presence of shared epitopes indicates striking conservation of centromere/kinetochore components throughout evolution.

A new cell line was established from a "neuroectodermal tumor of bone" affecting the right scapula of an 18-year-old man. The original neoplasm had dense proliferation of small round cells with abundant glycogen content and numerous Homer-Wright rosettes. The culture showed proliferation of small spindle cells with uniform oval nuclei and slender cytoplasmic processes. When the culture reached maximum density, rosette-like structures similar to those in the original tumor were formed. Under the influence of N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dibutyryl cAMP), the cultured cells expressed these rosette-like structures even in the lower cell concentration. Electron microscopy revealed that the cultured cells treated with dibutyryl cAMP contained high-density granules, well-developed microtubules, and abundant 10-nm filaments. By immunocytochemistry, neuron-specific enolase (NSE), and N-myc oncogene product were detected in the cultured cells as well as the original tumor. These results indicated the neuroectodermal origin of some of the small round cell tumors of bone.

We compared the organization of satellite DNA (stDNA) and its chromosomal allocation in Mus domesticus and in Mus musculus. The two stDNAs show similar restriction fragment profiles after digestion (probed with M. domesticus stDNA) with some endonucleases of which restriction sequences are present in the 230-240 bp repetitive unit of the M. domesticus stDNA. In contrast, EcoRI digestion reveals that M. musculus stDNA lacks most of the GAATTC restriction sites, particularly at the level of the half-monomer. The chromosome distribution of stDNA (revealed by an M. domesticus stDNA probe) shows different patterns in the M. domesticus and M. musculus karyotypes, with about 60% of M. domesticus stDNA retained in the M. musculus genome. It is particularly noteworthy that the pericentromeric regions of M. musculus chromosomes 1 and X are totally devoid of M. domesticus stDNA sequences. In both groups, the differences in energy transfer between the stDNA-bound fluorochromes Hoechst 33258 and propidium iodide suggest that AT-rich repeated sequences have a much more clustered array in the M. domesticus stDNA, as if they are organized in tandem repeats longer than those of M. musculus. Considering the data as a whole, it seems likely that the evolutionary paths of the two stDNAs diverged after the generation of the ancestral 230-240 bp stDNA repetitive unit through the amplification, in the M. domesticus genome, of a family repeat which included the EcoRI GAATTC restriction sequence.

Several nucleolar antigens are defined by human autoantibodies. These antigens can therefore be used to follow the fate of nucleolar components through mitosis when this major nuclear structure disintegrates and becomes reassembled in G1-phase. We found that fibrillarin leaves the nucleolus before complete breakdown of this structure and attaches to chromosomes before nuclear envelope breakdown. In mouse, fibrillarin attaches over the chromosomal surface except for the excluded centromeric region. The antigen is transported to the new nucleus via the chromosomes and is last seen on chromosomal surfaces facing the cytoplasm during nuclear envelope reformation. Lamin B reappears on the same chromosomal surfaces before the nucleolar antigen is removed and aggregates for new nucleolar reformation in G1-phase cells. From our observations, we postulate that the antigen acts in concert with other proteins as a nuclear envelope equivalent by forming a protective sheath around the chromosome, that it excludes larger molecules, and helps to separate the chromosomes, in addition to segregation of the ribonucleoprotein (RNP) back to the nucleus for nucleolar reconstruction. We also suggest that the selective retention of these antigens from certain areas on individual chromosomes together with specific lamin B attachment over these chromosomal surfaces allows for a nonrandom positioning of chromosomes in the nucleus.

During the last 10 years, we have cytogenetically analyzed at diagnosis bone marrow cells from a total of 94 children with acute leukemia. Of the 78 children with acute lymphatic leukemia (ALL), 53 (68%) had clonal acquired chromosome abnormalities; in the group with acute nonlymphatic leukemia (ANLL), the corresponding proportion was 13 out of 16 (81%). Among the cytogenetically abnormal ALL patients, the most numerous subset was the hyperdiploid cases with stemlines containing 51 or more chromosomes (26 of 53 abnormal cases; 49%). This is a clearly higher proportion than has been reported in large series from other centers. Deletions of 6q were present in 8 cases and rearrangements of 12p in 5. Of the 7 T-cell ALLs, 3 had translocations of the distal part of 7q, i.e., of the region where the beta T-cell receptor is encoded. Only 2 of 26 (8%) patients with leukemic stemlines with more than 50 chromosomes have relapsed; the remainder are still in first remission (mean observation time 42 months). This may be contrasted with 6 of 25 (24%) relapses among the cytogenetically normal (observation time 41 months), and 8 of 27 (30%) relapses among ALL patients with aberrations but with less than 51 chromosomes (observation time 26 months). Our results support the conclusion that the finding of a markedly hyperdiploid leukemia karyotype is indicative of good prognosis in ALL.

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.

Drosophila nasutoides has an extraordinary genome since 62% of its DNA resides in chromosome 4. This element mainly consists of constitutive heterochromatin which does not polytenize. Earlier studies of heterochromatin attributed little attention to the fact that "condensed" chromosomes often vary in condensation. This paper reports that chromosomes of the same complement display different degrees and kinetics of condensation. In D. nasutoides, even sex specific differences can be observed. The results of a comparative microphotometric study on neuroblast metaphases in both sexes revealed the following picture. The process of chromosome condensation is not restricted to mitotic prophase but continues into the metaphase. The mean condensation is not equal for all chromosomes. In the metaphase of the female, Feulgen density increases from the X chromosome, via 3 and 2, to chromosome 4. In the male, the order is X, 2, 3, Y, and 4. During the metaphase of the male, chromosomes condense with similar kinetics. In contrast, chromosomes of the female display asynchrony as monitored by area and length determinations. The X chromosomes of the female probably have enhanced shortening during prophase. This would explain the metaphase of the female where the X chromosomes shorten less than the autosomes, and why each of the X chromosomes is 15% shorter than the X chromosome in the metaphase of the male. Further differences were observed in the longitudinal and lateral compaction of the chromosomes in males and females. The sex chromosomes and chromosome 3 condense by shortening, while chromosome 2 and 4 preferentially reduce their diameter. The large amount of DNA engaged in heteropycnosis and the isochromosome nature allow the identification of chromosome 4 during interphase. At this stage, a new category of extreme DNA packaging was detected. The interphase density of chromosome 4 can exceed that of metaphase by a factor of up to 8. Two events account for this high degree of condensation: (1) the homologues are particularly associated due to somatic pairing and (2) the arms are further tightened as a result of pericentric folding. The features of the isochromosome suggest that the interaction of chromatids during interphase is essentially caused by specific DNA sequences. The data confirm that heteropycnosis not only interferes with gene expression but also strongly inhibits DNA synthesis in endocycles.

The molecular mechanisms of reversion in mammalian cells were studied utilizing the pZipGptNeo shuttle vector, with the bacterial gpt gene in the vector integrated into the chromosomal DNA of mouse cells. From mutant cell lines containing gpt genes with single base changes, revertants were selected for the reappearance of GPT activity. The copy number and expression of the gpt genes in such revertants were analyzed, and the GPT activity encoded by revertant genes in both mammalian cells and bacteria characterized. Revertants with wild-type amino acid sequence had, on average, the highest levels of GPT activity. Revertants with amino acid sequences different from the original mutants but not corresponding to wild-type had, on average, approximately half the level of GPT activity as wild-type revertants. Revertants that still contained the original mutation in the gpt gene had even lower levels of activity. These revertants were found to have amplified mutant gpt genes, which, when transferred into bacteria, were seen to encode for GPT polypeptides with partial enzymatic activity. A revertant in which the original mutation that destroyed the AUG translational start codon was retained but in which there was a secondary mutation upstream of the start codon also was characterized. The second mutation generated an in-frame CUG codon that apparently functioned as an alternative, upstream translational start codon.

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 total number of oocytes at different postmating time intervals (18-40 days) was determined in mice homozygous and heterozygous for different Robertsonian (Rb) translocations, of both laboratory and feral origin. The number of oocytes was lower in heterozygous than in homozygous mice throughout the period studied. Independently of the genetic background (i.e. laboratory or feral), structural heterozygosity had a progressive detrimental effect on oocyte numbers: open, or chain diakinetic configurations had a greater detrimental effect than close, or ring, configurations. The genetic background, however, affected the ovarian constitution in terms of the total number of germ cells, which are more numerous in laboratory than in feral mice. The kinetics of oogenesis seems to be faster in feral than in laboratory mice. At the light of the data here presented, and of those already available from the literature on male and female gametogenesis in conditions of structural heterozygosity, it appears that factors other than unsaturation of pairing sites or interference with pachytene X-chromosome inactivation have to be considered. In the wild, the reduced oocyte numbers in Rb heterozygous female can contribute to the retention of isolated populations in contact zones.

This review is an attempt to summarize all existing data on histone H1 and high mobility group proteins obtained with immunochemical methods. The following issues are treated consecutively: production of specific antisera to these protein groups, antigenic structure of the polypeptide chains, use of antibodies for the identification, the quantitative estimation and the study of the tissue- and species-specificity of the proteins. Special attention is devoted to the studies of the localization of the respective antigens in the cell, the nucleus, the chromosomes and the interphase chromatin. The use of specific antibodies for the elucidation of the role these proteins play in such basic cellular processes as proliferation and differentiation, replication and transcription is also discussed. It becomes clear that the use of immunochemical approaches in the study of specific chromatin proteins both at the level of the protein molecule and at the level of chromatin can be a powerful tool for the resolution of a number of specific problems. The field is very promising and will undoubtedly develop intensely in the nearest future.

DNA from sporozoites of Eimeria tenella was resolved by pulsed field gel electrophoresis into nine chromosomal bands. Some bands of this molecular karyotype contained more than one chromosome as determined by the relative intensity of both staining with ethidium bromide and hybridisation to an E. tenella telomeric probe. Haploid forms of the parasite must be presumed to contain at least 12 chromosomes. The two smallest chromosomes were about 1.1 and 1.4 megabases. Most chromosomes were in excess of 3 Mb with the largest over 5 Mb as determined by comparison with the co-migration of chromosomes from Schizosaccharomyces pombe. A 5S ribosomal gene probe hybridised to a single chromosomal band.

In situ selective digestion of mitotic chromosomes from three Drosophila species (virilis, hydei, and funebris), having a chromosome number 2n = 12, was achieved with AluI restriction endonuclease. The distribution of AluI-bands, revealing a heterogeneity within heterochromatin, was compared with that of quinacrine-bands observed in standard chromosomes. The results confirmed a species-specific AluI-banding pattern, heterochromatin being selectively digested in D. virilis and D. funebris, but unaffected by the enzyme in D. hydei. The overlapping of AluI-bands and Q-bands in D. virilis and in D. funebris is discussed, since these data seem to favour the hypothesis that the induction of AluI-bands does not depend only on the presence of enzyme targets, but also on a suitable conformation of the chromatin.

We describe the construction of two complete sets of small insert, complete digest DNA libraries for each of the 24 human chromosomal types by the National Laboratory Gene Library Project. Flow sorting was used to purify the chromosomes which provided the DNA for cloning. One set of libraries was cloned into the HindIII site of the lambda vector Charon 21A, and the other set was cloned into the EcoRI site of the same vector. Characterization information from both in-house experiments and user feedback is presented. These chromosome-specific libraries are available to the general scientific community from a repository at the American Type Culture Collection, Rockville, MD. The second phase of the project, the construction of large insert, partial digest libraries in both lambda and cosmid vectors, is underway.

The chromosomal complements of five species of mammals, Mus musculus (Linn.), Rattus rattus rattus (Linn.), Bandicota bengalensis (Gray and Hardw.), Funambulus pennanti (Wr.) and Lepus ruficaudatus (Geoff.) were elaborated. The karyotypes of all the species were compared. The diploid number (2n) in Mus is 40, Rattus 42, Bandicota 42, Funambulus 54 and Lepus 48. An extra large X-chromosome was observed in one population of male Rattus rattus rattus (Linn.), which might have been formed as a result of duplication of the terminal segments of both the arms of the chromosomes.

It has been suggested that the chromosome set of the Indian muntjac, Muntiacus muntjak vaginalis (female, 2n = 6; male, 2n = 7), evolved from small acrocentric chromosomes, such as those found in the complement of the Chinese muntjac, M. reevesi (2n = 46), by a series of tandem fusions and other rearrangements. The location of the highly conserved human telomeric sequence (TTAGGG)n in the metaphase chromosomes of M.m. vaginalis and its close relative, M. reevesi, was investigated by non-radioactive in situ hybridization. The (TTAGGG)n repeat was found adjacent to the centromeres in the short arm and at the telomeres in the long arm of M. reevesi acrocentric metaphase chromosomes. Tandem fusions present in the karyotype of M.m. vaginalis chromosomes were not reflected by interstitial signals of the telomere repeat, as these chromosomes displayed hybridization signals only at the ends of the chromatids. Mechanisms that might have played a role in the evolution of the reduced karyotype of the Indian muntjac are discussed.

Phylogenetic relationships within the Drosophila sulfurigaster species-complex, which belongs to the D. nasuta subgroup, were investigated on the basis of chromosomal constitution and morphology. D. pulaua is thought to be the most ancestral species, from which D. s. sulfurigaster and D. s. bilimbata derived in one branch and D. s. albostrigata and D. s. neonasuta in another branch.

A method for the cultivation of monolayer cell cultures on microslides in quadruple culture dishes together with a simple procedure for in situ chromosome preparation are described. The cells fixed to the slide can be stained according to standard procedures and analysed microscopically. The method is simple, rapid and reliable and provides many advantages especially for cytogenetic diagnostics with fibroblasts and amniotic fluid cells. It simplifies the performance of cytogenetic mutagenicity testing with primary cultures and permanent cell lines, e.g. the analysis of chromosome aberrations, sister chromatid exchanges (SCEs) and induced aneuploidy, as well as large-scale cytogenetic experiments.

The direct method of chromosomal preparations on chorionic villi biopsies has been applied to the cytogenetic study of 34 early spontaneous abortion products. These results were compared with those obtained through long term cultures performed simultaneously from the same samples. The success rate obtained through the direct method (86.2%) is superior to that of the long term culture (76.4%). However, the combined use of both techniques further improves the success rate (94.1%) and the reliability of the results. Among the 32 informative cases, 15 chromosomal aberrations (46.8%) have been detected. These data demonstrate the value of the direct method of karyotyping chorionic villi from spontaneous abortion products. However, considering the specific advantages of either method respectively, the authors recommend that both be used simultaneously for optimal diagnostic efficiency.

The position of a mouse DNA repeat located near the centromere of mouse chromosomes X, 11, 13, and 17 was examined in interphase nuclei of bone marrow and fibroblast cells by in situ hybridization of 3H- or biotin-labeled DNA probe 70-38. In most laboratory mouse strains this probe recognizes a single repeat cluster (DXWas70) close to the centromere of the mouse X chromosome. In a few mouse strains, a second locus (D11Was70, D13Was70, or D17Was70, depending on the mouse strain) is located near the centromere of an autosome. In interphase nuclei from mouse strains with the X-linked locus only, two distinct sites of hybridization were found in female mice and one in male mice. These two sites remained separated during the different phases of the cell cycle (G1, early S, late S, and G2) as demonstrated by in situ hybridization of the probe to flow-sorted nuclei. In interphase nuclei from mouse strains with both the X-linked locus and an autosomal locus, four distinct sites of hybridization were found in female mice and three in male mice. Further analysis of loci DXWas70 and D17Was70 showed that these loci were often located in the outer region of nuclei from bone marrow and fibroblast cells.

The risk of bearing a child with chromosomal defects increases once the mother reaches the age of 35; the principal indication for chromosomal examination of the fetus is that the pregnant woman is "older". Chromosomal examination of new-born babies is indicated if it is suspected that dysmorphic features may depend on cytogenetic deviation. Other indications include suspicion of so-called fragile X syndrome or rare congenital disorders such as Fanconi's anemia, Xeroderma pigmentosum etc. Chromosomal examination is routine today in investigation of suspected or verified hematological diseases. Nevertheless the possibilities of cytogenetic techniques have not yet been used to the full in diagnostics, assessment of prognoses, choice of therapy and follow-up of disease.

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.

Transposition of the abl gene was demonstrated in seven Philadelphia (Ph'-) negative patients with either chronic myeloid leukaemia (CML) or acute lymphocytic leukaemia (ALL) by chromosomal in situ hybridization. In six out of seven CML patients and one out of two ALL patients, a significant accumulation of abl hybridization grains was localized to chromosome 22. Hybridization with both abl and sis resulted in the consistent formation of double hybridization events on chromosome 22. Transposition of abl was not apparent in one patient with Ph'-negative CML and in one patient with Ph'-negative ALL. The data suggest that transposition of abl to chromosome 22 is a feature of a particular subgroup of Ph'-negative leukaemias.

Chromosomal in situ suppression (CISS)-hybridization of biotinylated phage DNA-library inserts from sorted human chromosomes was used to decorate chromosomes 1 and 7 specifically from pter to qter and to detect structural aberrations of these chromosomes in irradiated human peripheral lymphocytes. In addition, probe pUC1.77 was used to mark the 1q12 subregion in normal and aberrant chromosomes 1. Low LET radiation (60Co-gamma-rays; 1.17 and 1.33 MeV) of lymphocyte cultures was performed with various doses (D = 0, 2, 4, 8 Gy) 5 h after stimulation with phytohaemagglutinin. Irradiated cells were cultivated for an additional 67 h before Colcemid arrested metaphase spreads were obtained. Aberrations of the specifically stained chromosomes, such as deletions, dicentrics, and rings, were readily scored after in situ hybridization with either the 1q12 specific probe or DNA-library inserts. By the latter approach, translocations of the specifically stained chromosomes could also be reliably assessed. A linear increase of the percentage of specifically stained aberrant chromosomes was observed when plotted as a function of the square of the dose D. A particular advantage of this new approach is provided by the possibility to delineate numerical and structural chromosome aberrations directly in interphase nuclei. These results indicate that cytogenetic monitoring of ionizing radiation may be considerably facilitated by CISS-hybridization.

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.

In these studies a polyclonal antiserum elicited against a carcinogen-DNA adduct was used to explore the localization of DNA adducts in metaphase chromosomes of cultured cells. Morphological visualization of the adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) in Chinese hamster ovary (CHO) cells exposed to the direct-acting carcinogen N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) was accomplished by indirect immunofluorescence with an anti-G-C8-AF antiserum. At the same time the pattern of chromosomal DNA replication was determined by replicative incorporation of bromodeoxyuridine (BrdUrd) and chromosomal staining with anti-BrdUrd. Visualization of DNA in chromosomes was accomplished with Hoechst 33258 dye. When synchronized CHO cells were exposed to N-Ac-AAF for 0.5 h during early S phase, the chromosomal pattern of dG-C8-AF adduct formation was not random. Metaphase chromosome spreads from cells exposed to N-Ac-AAF in different experiments contained certain chromosome regions that had a consistently high adduct concentration. The regions of high DNA damage corresponded to the regions active in DNA synthesis when BrdUrd and the carcinogen were given simultaneously in early S phase. In addition, the patterns of high adduct concentration and replicative synthesis shifted when the carcinogen and BrdUrd were given simultaneously during late S phase. Thus, the stage of cell cycle in which adducts are induced is an important factor in the specific location of the highest concentrations of this type of DNA lesion.

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).

In order to score for the fragile X syndrome, blood samples are prepared with absorption stain labeling by in situ hybridisation of the X chromosome centromeres. Metaphases are located, digitised at high resolution, and segmented fully automatically. A three stage adaptive classification scheme for labeled X chromosomes is then applied. This consists of a simple box classifier to identify plausible X and false positive X chromosomes, followed by a quadratic discriminant classifier that is re-trained for each sample. The modal number of X chromosomes is then determined for each sample and used to refine the classification. A simple fragile site detector is applied to the distal portion of the detected X chromosome long arms. From the results we estimate computer and operator time requirements for a screening system in which the operator reviews only the apparently fragile X chromosomes detected by the computer.

The viral DNA sequences in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV-1) virions, by full-length linear BPV-1 DNA, or by a defined transforming subgenomic DNA segment of BPV-1 were examined by reassociation kinetics and blot hybridization. In all cases, the transformed cells contained multiple copies of BPV-1 DNA, present exclusively as supercoiled or nicked circular extrachromosomal molecules or as a slowly migrating complex of circular viral DNA molecules. In the transformed cell lines established from cells transfected with full-length linear BPV-1 DNA, there was recircularization of the input DNA which in some cases resulted in the loss of the restriction site used in the linearization of the DNA. In the transformed cell lines established with the defined subgenomic segment there was circularization of the DNA accompanied by the acquisition of new sequences or duplication and rearrangement of the BPV-1 sequences. In contrast to other well-studied virus transformation systems, no integration of the BPV-1 genome into the host chromosome could be detected under conditions sensitive enough to detect 0.1-0.2 viral genome equivalent. It was concluded that maintenance of transformation may be mediated by nonintegrated viral DNA.

Typhlonectes compressicauda has a diploid number of 28. Its karyotype, when compared to that of other caecilians, suggests some discordance in the hypothesized model of chromosome reduction in the evolution of amphibian lineages.

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.

The effect of 1 microM dibutyril cAMP (cAMP media) on the chromosomal normality of in vitro fertilized squirrel monkey oocytes was examined. A total of 877 oocytes were collected laparoscopically 15-16 h after hCG injection from hormonally-induced follicles of 65 squirrel monkeys. Of these, 330 (37.6%) were prepared between 6 and 30 h after insemination and analyzed. Sperm penetration and the presence of a swollen sperm head in the ooplasm were observed by 6 h, and the nucleate stage by 10 h. Addition of 1 microM dbcAMP did not affect the oocyte maturation rate, however the in vitro fertilization rate was increased 26.5% over controls (46.3 vs. 36.6%). The first cleavage metaphase was found at 16 h after insemination. Between 24 and 30 h post-insemination, 76.6% of fertilized oocytes had reached the first cleavage metaphase. An incidence of triploidy (including dispermy) of 11.7% was observed. After chromosomal analysis of metaphase I, univalents were observed in 16.7% of the cases. Aneuploidy (8.9%) was also found in metaphase II. Of the first cleavage metaphases, 88.0% had the diploid number of chromosomes. Aneuploidy occurred 12.0% of the time and 5 cases of triploidies were observed.

A method is described for visualising chromosome-mediated gene transfer (CMGT) by detecting chromosomes labelled with bromodeoxyuridine (BrdU) using a monoclonal antibody to BrdU. In this experiment, the CCRF-CEM T cell line was grown in the presence of BrdU and the labelled chromosomes were isolated and transfected into human embryonic fibroblasts. Uptake and retention of chromosomes were compared for transfection with either PEG or DMSO treatments. Following transfection the labelled chromosomes could be visualised in recipient cells using a monoclonal antibody to BrdU, followed by immunoperoxidase staining. Chromosome uptake into cells was similar for both DMSO and PEG treatments and was a relatively frequent event; about 1 in 5 recipient cells had labelled material present. This technique can be used to assess the technical aspects of the earliest stages of chromosome-mediated gene transfer.