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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Simplified "overnight" and longer term primary culture techniques are described for obtaining chromosome preparations from tiny chorionic villi samples (5-15 mg).

The immunological accessibility of the nucleosomal core protein H3 and non-histone protein, HMG-1, was studied in transcriptionally active regions of Chironomus thummi polytene chromosomes. Chromosomal loci were decondensed by incubating isolated salivary glands in various salt solutions or hemolymph. Indirect immunofluorescence studies on these chromosomes using anti-sera to histone H3 revealed that the puffed regions were depleted of fluorescence. The lack of fluorescence could be correlated with the degree of puffing and the level of transcriptional activity. The puffed regions fluoresce after anti-H3 addition if the chromosomes are not cross-linked with formaldehyde, and if prior to the addition of antibodies the chromosomes are treated with 45% acetic acid. We conclude that, whereas histone H3 is present in the puffed regions, its antigenic determinants are sterically hindered by components which are extractable by 45% acetic acid. On the other hand, the antigenic determinants of protein HMG-1 are always available to antibody binding in puffed regions, as well as other chromosomal areas.

A methotrexate (MTX)-resistant murine lymphoblastoid cell line has been obtained by serial passage in increasing concentrations of MTX which is greater than 100,000-fold resistant to MTX (L5178YR) and has dihydrofolate reductase (DHFR) levels 300-fold higher than the parental line. The L5178YR cell line synthesizes approximately 10-11% of its total soluble cell protein as DHFR regardless of growth phase, as measured by direct immunoprecipitation with a monospecific antiserum. Molecular hybridization of a purified [3H]DNA probe complimentary to DHFR specific mRNA with cellular DNA and RNA indicates that DHFR coding sequences are elevated several hundred fold in both nucleic acid species in the mutant cell line. Giemsa-banding studies of the diploid mutant line indicate the presence of a large homogeneously staining region on chromosome No. 2. In situ molecular hybridization studies indicate that the DHFR genes are localized in this homogeneously staining region. The homogeneously staining region probably consists of tandom repeats of a basic segment approximately 800 kilo base pairs long.

Three hundred young Dutch Friesian and Holstein-Friesian bulls, kept in the Central Opfokstation (Central Breeding Station) in Terwispel (Friesland), were studied cytogenetically, using conventional staining methods. Structural chromosome aberrations were not observed. Four animals showed XX/XY-chimerism in the lymphocytes, probably caused by the interchange of haemopoietic cells between the male and its female co-twin by placental vascular anastomosis. The number of (iso)chromatid gaps in 25 metaphase plates varied from 0 to 4. Chromosome breaks were not observed. The chimeric bulls and those with (iso)chromatid gaps were not found to show significantly reduced fertility rates.

Those non-histone chromosomal proteins which are easily extractible from chick erythrocytes differ substantially from proteins similarly extracted from other tissues of various species. Although a protein P1 was isolated along with histone H1 by extraction of calf thymus chromatin with HC1O4, the same procedure did not extract this protein from chick erythrocyte chromatin of either normal or regenerating blood. Likewise , non-histone proteins extracted with 0.35 M NaCl from calf thymus differed from those of normal chick erythrocytes, which were qualitatively identical but quantitatively inferior to those of regenerating blood. The major protein of about 25 000 molecular weight, totally extracted with 0.35 M NaCl from calf thymus, was not found in chick erythrocyte chromatin, but rather another major protein of about 35 000 molecular weight was partially extracted from erythrocytes.

SV40 chromosomes capable of continued RNA synthesis in vitro have been extracted from infected cells in solutions of very low ionic strength (5 mM HEPES, 0.25 mM MgCl2). The RNA made is of high molecular weight, and synthesis is sensitive to alpha-amanitin. More RNA is made than from previously described (high salt-extracted) transcription complexes. Transcribing SV40 chromosomes sediment at a rate intermediate between replicating and mature chromosomes, and have a higher ratio of protein to nucleic acid than either. They retain proteins that are lost during exposure to high salt, and might prove valuable in identifying proteins involved in SV40 transcription.

A technique is described by which unstained metaphases may be observed using phase contrast microscopy. The cells are fixed on a cover slip which then is turned over and onto a slide, leaving a thin air layer sandwiched between. Observation in phase contrast shows metaphases comparable to those observed after Giemsa staining.

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.