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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.

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.

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.

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).

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.

We have injected circular DNA, labeled with 32P at a single restriction site, into germinal vesicles of Xenopus laevis oocytes in order to study the nucleosome arrangement on the assembled minichromosomes. Two types of genes were used in these studies, the somatic 5 S RNA gene unit of Xenopus borealis and the histone gene unit of Drosophila melanogaster. We find that injections of labeled DNA alone, at 1 ng DNA per oocyte, results in irregularly spaced nucleosomes and partially supercoiled DNA molecules. However, perfectly spaced nucleosomes are assembled and fully supercoiled DNA is recovered if 5 to 20 nanograms of cold vector DNA is coinjected with the labeled DNA. At the optimum chromatin assembly conditions, the nucleosomes are perfectly spaced with a 180 base-pair periodicity, but they are randomly positioned on the DNA. The assembly of a periodic chromatin structure is accompanied by a dramatic enhancement in the expression of the injected 5 S RNA gene.

Mapping of the MHC in swine (SLA) was achieved by direct in situ hybridization to chromosome preparations. We took advantage of the fact that the cDNA probe coding for class I HLA-B7 antigen cross-hybridizes with swine genomic DNA. By nick-translation, 35S nucleotides were incorporated to a specific activity of 2,7 10(7) cpm/ug. Analysis of 91 randomly selected labeled metaphases revealed highly significant labeling on chromosome 7. The SLA complex is most probably located at the proximal half of the long arm, as indicated in families carrying a modified chromosome 7 and heterozygous for SLA. The abnormal chromosome was always inherited with a specific haplotype whereas the other parental haplotypes were found in association with the normal 7.

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.

Cytogenetic changes, namely numerical (aneuploidy) and morphologic chromosome abnormalities including markers, clearly indicate the presence of malignant cells. To compare the utility of chromosome and cytogenetic analysis, the idiopathic pleural effusions of 60 patients were subjected to a double-blind analysis. A fluid was considered "positive" for malignancy if there was a marker chromosome, or if 10 percent of metaphases were hyperdiploid. Of the 33 neoplastic effusion, 30 (91 percent) were diagnosed "positive" by chromosome analysis whereas only 21 (64 percent) were classified "positive" or "suggestive positive" by cytology. All 27 benign effusions were correctly diagnosed by cytology; however, two were labelled "positive" by chromosome analysis. Combining the results of cytologic and chromosome analysis did not increase the number of "positive" diagnoses.

Metaphase chromosomes and interphase nuclei have been isolated by two different procedures, using either acidic or alkaline media. The morphological integrity of the isolated material has been monitored by electron microscopy. In the acidic procedure the thickness of the chromosome fibers is reduced but the chromosome gross structure is preserved. However, electrophoretic analysis indicates complete disappearance of histones H1 and partial loss of H2A and H2B. No significant modification of acid-soluble proteins isolated from chromosomes or nuclei was observed after 5-BrdU incorporation for 72 hours.

A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.

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.

Mouse-human heterohybrids (M X H) were constructed and compared with other cell lines (human or mouse) as parental cells to obtain hybrids secreting human monoclonal antibody (MoAb). One of the M X H lines, HM-5, was far superior to the others and useful for establishing hybrids secreting human MoAb. Using HM-5 as a parental cell line, we have obtained 2 hybrids secreting human anti-tetanus toxoid MoAb with neutralizing activity and a hybrid secreting human anti-hepatitis B virus surface antigen (HBsAg) MoAb which recognizes the a-determinant of HBsAg.

A dominant sex-linked gene, K, regulates slow feathering (SF), whereas a recessive allele, k+, determines rapid feathering (RF) in chickens. This trait provides a convenient and inexpensive approach to gender identification of White Leghorn (WL) chicks at hatch, i.e., in a sex-linked mating using k+/k+ males mated with K/- females, the K/k+ male chicks are SF, and the k+/- females are RF. However, in many WL strains, female progeny of SF dams produce fewer eggs and have higher mortality than progeny of RF dams. This loss in productivity has been attributed to higher infection and shedding rates for leukosis viruses (ALV) in SF than in RF dam lines. Because infectious endogenous viruses (EV) can induce immunological tolerance to ALV, we examined the expression and distribution of ev genes in SF and RF siblings from heterozygous K/k+ sires and k+/- dams. Infectious ALV and EV were detected by cocultivation of frozen heparinized blood cells on selected chick embryo fibroblasts and culture supernatants were tested for viral antigen by enzyme-linked immunosorbent assay tests. Specific ev genes were identified as restriction fragment length polymorphisms after hybridization with a recombinant plasmid containing the complete genome of a Rous-associated virus. It was concluded that ev21 and K genes are tightly linked because, in different WL crosses, all SF chicks inherited ev21 but RF siblings uniformly lacked ev21. Alternatively, the K gene in WL may be a mutation resulting from the insertion of ev21 in the k+ gene. The SF chicks which harbor ev21 expressed infectious EV21; evidence that EV21 may influence susceptibility to ALV is presented.

An increased synthesis of fetal hemoglobin in adult life is a common feature of the genetically determined severe disorders like beta thalassemia and sickle cell anemia. A continued synthesis of fetal hemoglobin in adults is also characteristic of clinical or subclinical syndromes like respectively delta beta thalassemia or hereditary persistence of fetal hemoglobin (HPFH). These disorders are highly heterogeneous with respect to their molecular defects as well as to the composition of Hb F. We report here a novel case of hereditary persistence of fetal hemoglobin in heterozygous state discovered by chance, in a young perfectly healthy french man. The gamma chain of his fetal hemoglobin was almost entirely composed of G gamma chains. Molecular analysis of the DNA revealed the existence of triplicated gamma genes on one chromosome with the genotype arrangement of G gamma-G gamma-A gamma. A polymorphic Xmn I restriction site (at position -158 5' to the cap site) was present in 5' of both of these G gamma genes. The presence of this site in front of G gamma gene had previously been shown to be associated both with high G gamma phenotype constitutively and also with high fetal hemoglobin level only in case of anemic stress. In the absence of any anemic stress in this individual, the constitutive increase of both fetal hemoglobin and G gamma chains could be due to the presence of a chromosome with triplicated arrangement of gamma genes. The classical triplication (G gamma-A gamma-G gamma-A gamma) does not result in HPFH phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

The location and dynamics of small nuclear ribonucleoproteins (snRNPs) were studied in salivary gland polytene chromosomes of Chironomus tentans by immunofluorescence with specific snRNP antibodies. Monoclonal antibody against the snRNP Sm antigens reacted at all sites of transcription (puffs and Balbiani rings). The amount of snRNP immunofluorescence was strictly dependent on transcription, increasing in parallel with gene activation and decreasing upon repression. Identical patterns of localization and transcriptional dependence were observed with antibodies specific for U1 or U2 snRNPs. These latter results show that the involvement of U1 and U2 snRNPs in transcription-related processes involves a high proportion, rather than small subsets, of active gene loci. In addition, the colocalization of U1 and U2 snRNPs at loci known to contain only one messenger RNA transcription unit (e.g. Balbiani ring 2) raises the possibility that both of these snRNPs interact with the same transcript. Finally, the lack of immunofluorescence at repressed loci indicates that snRNPs are not structural components of the chromatin (DNP) fiber, and also shows that unused snRNPs are not stored in chromatin. These latter points, and the growing evidence for the involvement of U1 snRNP in splicing, suggest that nascent pre-mRNA is the major chromosomal binding site for snRNPs.

This work describes the development of a method for fractionating the total nuclear proteins of Chinese hamster cells (line CHO) by high-performance liquid chromatography of whole nuclei or chromosomes. Nuclei or chromosomes were dissolved for 2 h in 6 M guanidine hydrochloride containing 0.2% trifluoroacetic acid (TFA). Residual particulates were removed by centrifugation, and a sample of the solution was passed through a Bio-Sil TSK guard column, followed by a Bio-Sil TSK 400 column, equilibrated with water containing 0.2% TFA. These columns fractionated the sample by adsorbing the DNA, passing the proteins near the exclusion limit, passing the guanidine at the inclusion limit, and passing an unidentified non-protein/non-DNA material after the inclusion limit. The protein peaks from the TSK columns were collected directly on a muBondapak CN reversed-phase column. The protein fractions were then eluted from the CN column with an acetonitrile gradient containing 0.2% TFA. Three other reversed-phase columns were examined for use with the TSK columns. The muBondapak C18 Radial-Pak column produced the best resolution of histone variants, while the Zorbax C8 column produced a better resolution of the non-histone proteins. The Nova-Pak C18 Radial-Pak column was found to be unsatisfactory for both classes of nucleoproteins.

SCI is a prominent, 170,000 Mr, non-histone protein of HeLa metaphase chromosomes. This protein binds DNA and was previously identified as one of the major structural components of the residual scaffold structure obtained by differential protein extraction from isolated chromosomes. The metaphase scaffold maintains chromosomal DNA in an organized, looped conformation. We have prepared a polyclonal antibody against the SC1 protein. Immunolocalization studies by both fluorescence and electron microscopy allowed identification of the scaffold structure in gently expanded chromosomes. The micrographs show an immunopositive reaction going through the kinetochore along a central, axial region that extends the length of each chromatid. Some micrographs of histone-depleted chromosomes provide evidence of the substructural organization of the scaffold; the scaffold appears to consist of an assembly of foci, which in places form a zig-zag or coiled arrangement. We present several lines of evidence that establish the identity of SC1 as topoisomerase II. Considering the enzymic nature of this protein, it is remarkable that it represents 1% to 2% of the total mitotic chromosomal protein. About 60% to 80% of topoisomerase II partitions into the scaffold structure as prepared from isolated chromosomes, and we find approximately three copies per average 70,000-base loop. This supports the proposed structural role of the scaffold in the organization of the mitotic chromosome. The dual enzymic and apparent structural function of topoisomerase II (SC1) and its location at or near the base of chromatin loops allows speculation as to its involvement in the long-range control of chromatin structure.

A novel solid phase radioimmunoassay using nitrocellulose filters, is described. The method is of particular value in screening hybridoma cultures for monoclonal antibodies against chromosomal protein. Also it permits detection of antibodies directed against epitopes stable to SDS denaturation.

Previously we reported the existence of a highly polymorphic satellite, deca-satellite, in the African green monkey genome; deca-satellite probe anneals to complex sets of repeated restriction endonuclease fragments that differ from individual to individual in the monkey population. Here we present experiments aimed at clarifying the structure and organization of deca-satellite sequences and investigating the mechanisms that generate the polymorphisms. Deca-satellite represents less than 1% of the monkey genome but the percentage varies from one monkey to another. The core sequence 5'-C-C-G-G within the ten base-pair deca-satellite repeat unit is well conserved and the central 5'-C-G is sometimes but not always methylated. Restriction endonuclease analysis with BamHI and EcoRI defines separate satellite domains that have evolved in an independent manner. In situ hybridization shows deca-satellite to be located at the centromeric regions of some but not all monkey chromosomes. This location is independently confirmed by a high frequency, in monkey libraries, of segments containing junctions between deca-satellite and alpha-satellite, the main monkey centromeric satellite. The total number of metaphase chromosomes that show centromeric grains after in situ hybridization with a deca-satellite probe varies from one monkey to another. Moreover, in situ hybridization to endoreduplicated diplochromosomes showed that deca-satellite is occasionally distributed asymmetrically on one or the other of the two pairs of sister chromatids in one diplochromosome. This indicates that major reorganization of the satellite can occur frequently in somatic cells. We discuss several possible mechanisms by which deca-satellite sequences could be either amplified or deleted during a single replicative cycle. Also, on the basis of the marked fluidity of deca-satellite abundance and organization and other well-known attributes of centromeric satellites, we suggest that the existence and maintenance of centromeric satellite rests on the role of the tandem repeats themselves and not on any particular nucleotide sequence, repeat length or organization.

Chironomus tentans fourth instar larvae were awakened from oligopause (diapause) and treated with pilocarpine to stimulate Balbiani ring transcriptional activity. Salivary glands were fixed, sectioned and examined by conventional and stereo-electron microscopy. Balbiani ring RNP lateral fiber densities were measured in well-formed regions with clear Balbiani ring granules arranged along chromatin transcription axes. Despite an up to ten-fold range in Balbiani ring transcriptional activity [10, 12, 14], we observed no significant difference in lateral fiber densities. These data are discussed in terms of the variety of mechanisms for modulating overall transcriptional activity, including recruitment of parallel transcription units in the polytene chromosome. The lack of other major ultrastructural changes at the nuclear envelope and within the domains of tubular rough endoplasmic reticulum argue strongly against a major dismantling of subcellular structure during the induced oligopause.

The karyotypes of Cebus capucinus and C. nigrivittatus (Primates, Platyrrhini) are compared after applying several banding techniques. The chromosomes have abundant intercallary heterochromatin which can be stained by R-, T- and C-band techniques and which are late replicating. The X chromosome resembles that of man and of numerous primates. However, the late replicating pattern of the X in female lymphocytes resembles that of the late replicating X of human fibroblasts rather than of human lymphocytes. Banding patterns of certain chromosomes appear analogous in Cebus and Cattarhini, including Man.

We have isolated, purified and characterized a 42-kDa phosphoprotein which has been found to be preferentially associated with active gene loci of salivary gland cells of Chironomus tentans. The rapidly phosphorylated form of this protein could be extracted with 0.2 M NaCl. Chromatographic analysis by gel filtration revealed that a significant fraction of labelled 42-kDa polypeptide elutes with an apparent molecular mass of 150 to 200 kDa. The result suggests that a portion of the phosphorylated 42-kDa polypeptide in native state forms a multisubunit protein complex consisting of rapidly phosphorylated 42-kDa polypeptide chains alone.

The relationship between the quantity of silver-stained interphasic nucleolar organizer regions (NORs) and nuclear synthetic activity, caryotype, and growth rate was studied in two established neuroblastoma cell lines (CHP 212 and HTB 10). Statistical analysis of silver-stained NORs revealed four times as many in CHP 212 cells compared with HTB 10 cells. No difference was observed in the ribosomal RNA synthesis between the two cell lines. The caryotype index was 1.2 for CHP 212 and 1.0 for HTB 10 cells. The number of chromosomes carrying NORs and the quantity of ribosomal genes was found to be the same for the two cell lines. Doubling time of CHP 212 cells was 20 hours compared with 54 hours for HTB 10 cells. In CHP 212 cells bindering of cell duplication by serum deprivation induced a progressive lowering (calculated at 48, 72, and 96 hours) of the quantity of silver-stained interphasic NORs. Recovery of duplication by new serum addition induced, after 24 hours, an increase of the quantity of silver-stained interphasic NORs up to control levels. In the light of available data, these results indicate that the quantity of interphasic NORs is strictly correlated only to the growth rate of the cell.