Early replication banding reveals a strongly conserved functional pattern in mammalian chromosomes

One of the best documented autosomal linkage associations in man is on chromosome 1p and in the mouse on chromosome 4. On mitotic chromosomes this genetic homology is shown more clearly by early replication banding (RBG; induced by incorporation of 5bromodeoxyuridine (BrdU) in the second half of the S phase) than by structural banding (induced on prefixed chromosomes by denaturation, RFA, or trypsin, GTG). To analyse this phenomenon in more detail, 11 chromosomal regions in man and the domestic cat with known genetic homology were compared. In four chromosome pairs RBG and GTG banding show the same degree of homology. In seven chromosome pairs the homology is more pronounced by RBG than by GTG banding. RFA banding does not reveal the same extent of homology as does RBG banding. These results clearly show a difference between the structural banding pattern, RFA and GTG, and the replication banding pattern, RBG. The following conclusions can be drawn: in chromosomal regions with homologous functions the DNA replicates in the same temporal order. Early replication banding (RBG) reveals a functional pattern in these regions which has been more strongly preserved during evolution than the underlying chromosomal DNA. Differences in chromosomal banding are most prominent in the GTG banding pattern, whereas similarities are most apparent in the RBG banding pattern.     

Maternal or fetal origin of rhesus monkey (Macaca mulatta) amniotic fluid leukocytes can be identified by polymerase chain reaction using the zinc finger Y gene.

Leukocytes can be found in substantial numbers within the intrauterine tissues and amniotic fluid of women, and play a central role in the pathophysiology of infection-related preterm labor by their production of proinflammatory mediators. It remains unclear whether these leukocytes represent a fetal immune response, a maternal response, or a combination of the two. The objective of this study was to develop a test in the rhesus monkey (Macaca mulatta) suitable for determining the percentage of male fetal cells present in a population of leukocytes recovered from blood or amniotic fluid. We found inadequate specificity for rhesus monkey cells using commercial human Y-chromosome paint kits (fluorescence in situ hybridization (FISH)). Human-specific primers for the repetitive Y chromosome DYZ-1 locus employed in the polymerase chain reaction (PCR) produced an unacceptable percentage of false positives. However, we successfully developed a PCR-based test using rhesus-specific primers for the zinc finger Y (ZFY) locus. Densitometry of PCR products from known ratios of male and female adult peripheral leukocytes generated a linear standard curve which provided quantitative results and required only 400 cells per sample. The rhesus beta globin (RBG) gene served as an internal control. The PCR test correctly discriminated the sex of peripheral leukocytes in 20 adult males, 20 adult females, two male fetuses, and one female fetus. Serial samples of amniotic fluid from four chronically catheterized rhesus monkeys bearing male fetuses were used to confirm the utility of this assay for quantifying fetal cells in amniotic fluid. In conclusion, we have developed a PCR test which is suitable for distinguishing male from female cells in adult and fetal blood and in amniotic fluid, which lends itself to a variety of diagnostic and biologic applications in the rhesus monkey and potentially in other nonhuman primates.     

Description of the mallard duck (Anas platyrhynchos) karyotype

The karyotype of the mallard duck, Anas platyrhynchos, was characterised on the basis of R and C bands. Chromosomal preparations obtained from in vitro blood lymphocyte cultures were RBG- and CBG-stained. The structures of nine and 14 pairs of chromosomes were analysed by the RBG and CBG chromosome banding techniques, respectively. The location of R bands, as well as the size and arrangement of constitutive heterochromatin blocks were determined. Ideograms of R and C banded patterns of the analysed chromosomes were drawn. The morphological makeup of the analysed chromosomes was assessed.     

Cell morphology in long-term cultures of normal and abnormal amniotic fluids

Summary The cell morphology of long-term cultures of amniotic fluid cells from 10 fetuses with a neural tube defect (NTD) and three with omphalocele was examined and compared to 30 long-term cultures of normal amniotic fluids as well as a long-term culture of human fetal brain. Cultures from the amniotic fluids of the fetuses with NTD and omphalocele showed cells with the same general characteristics as normal amniotic fluid cells. However, the cultures of amniotic fluid cells from NTD pregnancies had an additional cell type also seen in fetal brain culture. This was a neuroblast-like cell, with small rounded refractile morphology and long branching processes forming clusters of varying sizes which lay on top of large flat cells. These neuroblast-like cells diminished in number with time in culture and were not present in subcultures. Their possible neuronal origin is discussed.     

Identification of cells from fetal bladder epithelium in human amniotic fluid

Summary Cultured human amniotic fluid cells consist of five different types of cytokeratin-positive epithelial cells, E-1 to E-5, differing by their size, growth morphology, and cytokeratin pattern, according to our earlier investigations. Using anticytokeratin antibodies in indirect immunofluorescence (IIF) microscopy, we show in this study that cultured urine cells contain four of the cell types found in amniotic fluid. In addition, we used two urothelium-specific antibodies, anti-UMA and anti-Las-86, in combination with cytokeratin antibodies to distinguish urothelium-derived cells in amniotic fluid and urine cell cultures. Two of the epithelial cell types were found to express urothelial antigens and thus to originate from the transitional bladder epithelium. These cells were found in 26 of the 33 amniotic fluid cell cultures and in nine of the ten urine cell cultures.     

Bovine fetal fluid cells in vitro: Fate and fetal sex prediction accuracy

Summary The in vitro fate of bovine fetal fluid cells and the efficiency of fetal sex prediction from cultures of these cells are studied using aspirates from live animals and pregnant uteri collected from the slaughterhouse. Over 70% of bovine amniotic fluid samples aspirated from pregnant uteri at the time of slaughter yielded cultures adequate for chromosome analysis, whereas only 10% of allantoic fluid samples produced growth of cells satisfactory for chromosome analysis. Fetal sexing accuracy was 100% in all samples studied. Seven readily recognizable cell types were noted in cultures of fetal fluid cells obtained at various stages of gestation. In a majority of cases, the in vitro morphology of cells from both fetal cavities was similar to that observed in primary human amniotic fluid cell cultures.     

Bovine fetal fluid cells in vitro: fate and fetal sex prediction accuracy

The in vitro fate of bovine fetal fluid cells and the efficiency of fetal sex predication from cultures of these cells are studied using aspirates from live animals and pregnant uteri collected from the slaughterhouse. Over 70 percent of bovine amniotic fluid samples aspirated from pregnant uteri at the time of slaughter yielded cultures adequate for chromosome analysis whereas only 10 percent of allantoic fluid samples produced growth of cells satisfactory for chromosome analysis. Fetal sexing accuracy was 100 percent in all samples studied. Seven readily recognizable cell types were noted in cultures of fetal fluid cells obtained at various stages of gestation. In a majority of cases, the in vitro morphology of cells from both fetal cavities was similar to that observed in primary human amniotic fluid cell cultures.     

Differentiation in human amniotic fluid cell cultures: chorionic gonadotropin production.

Two of the distinguishable cell classes subcultured from human amniotic fluid were examined for their capability to produce human chorionic gonadotropin (hCG) as determined by radioimmunoassay. The class that predominates in most cultures used for prenatal genetic diagnosis, previously termed AF (for amniotic fluid), secretes hCG into the culture medium. Dermal fibroblasts do not, nor does another type of cultured cell from amniotic fluid, previously termed F because of a resemblance to fibroblasts. Primary AF cultures produce more hCG than do subcultures. Evidence that this hormone is intact hCG is provided by its immunoreactivity with antisera raised against the beta-subunit and against the intact molecule of hCG. Furthermore, a dose-response curve for hormone in culture medium is parallel to that of highly purified intact hCG. It is postulated that AF cultures are derived from fetal membranes and retain properties of trophoblast.     

BrdU related direct revelation of typical dynamic R- and G-banding with the use of monoclonal anti-BrdU antibody.

Pulse 5-bromodeoxyuridine (5-BrdU) incorporation during the last S-phase is known to produce R- or G-banded chromosomes after photolysis-plus-Giemsa (FPG) staining. The authors applied an immunological staining with monoclonal anti-BrdU antibody instead of the FPG protocol. The results offered banded chromosomes with an immunological typical R-banding (RBI) on the GBG cultivated cells (early pulse incorporation), and an immunological G-banding (GBI) on the RBG cultivated ones (late pulse incorporation). After a further FPG protocol following an immunological treatment, an inverted banding pattern became evident whereas a faint immunological staining remained. Thus the method superimposed a GBG-banding on the RBI-staining or a RBG on the GBI one. This allows a rapid and easy R and G double chromosomal identification on the same metaphase cell, using first the immunological banding then the classical FPG staining. The method allows a reproducible dynamic G-banding with an easy monitored late 5-BrdU pulse incorporation specially attractive in spontaneous dividing cells from bone marrow. This dynamic G-banding protocol should be extended to chorionic villi and malignant cells. Our data are in agreement with a connection between dynamic banding and chromosomal portions containing or not BrdU. The lack of an immunological staining after the FPG protocol has been noticed and assume the photolysis degradation-elution of the DNA in BrdU-substituted areas.     

Differentiation in human amniotic fluid cell cultures: Chorionic gonadotropin production

Summary Two of the distinguishable cell classes subcultured from human amniotic fluid were examined for their capability to produce human chorionic gonadotropin (hCG) as determined by radioimmunoassay. The class that predominates in most cultures used for prenatal genetic diagnosis, previously termed AF (for amniotic fluid), secretes hCG into the culture medium. Dermal fibroblasts do not, nor does another type of cultured cell from amniotic fluid, previously termed F because of a resemblance to fibroblasts. Primary AF cultures produce more hCG than do subcultures. Evidence that this hormone is intact hCG is provided by its immunoreactivity with antisera raised against the β-subunit and against the intact molecule of hCG. Furthermore, a dose-response curve for hormone in culture medium is parallel to that of highly purified intact hCG. It is postulated that AF cultures are derived from fetal membranes and retain properties of trophoblast. Research supported by Grand HD 11379 from the National Institutes of Health.     

Heterogeneity for adenosine deaminase deficiency: Expression of the enzyme in cultured skin fibroblasts and amniotic fluid cells.

Adenosine deaminase (ADA) could be quantitated and the isozyme pattern characterized in cultured amniotic fluid cells. In 20 amniotic fluid cell cultures the mean specific activity was 14.3 U/g protein +/- 6.7 (SD) and compared favorably with values of 14.6 U/g protein +/- 6.8 (SD) observed in 26 cultures of skin fibroblasts. In cultures of skin fibroblasts established from two obligate heterozygotes for ADA deficiency, the specific activity of ADA was 7.0 and 7.7 U/g protein. The ADA isozyme pattern that existed in cultures of amniotic fluid cells was the same as that observed in cultured skin fibroblasts. This identification of the same apparent enzyme may permit the prenatal diagnosis of that form of combined immunodeficiency disease caused by ADA deficiency. Residual enzyme activity of less than 1% and 10% of the mean of normal fibroblasts could be measured in cultured fibroblasts from two unrelated children with ADA deficiency and combined immunodeficiency disease. The tissue-specific enzyme from cultured skin fibroblasts from the child with 10% residual activity had a faster electrophoretic mobility and greater heat stability than normal ADA. This enzymatic evidence indicates that at least two mutant alleles exist at the locus for ADA which predispose to combined immunodeficiency disease when present in the homozygous state.     

Inhibition of heterochromatin condensation of human Y chromosome by distamycin-A

Distamycin-A, an oligopeptide antibiotic with a N-methylpyrrole ring system and propionamide side chain, preferentially forms stable bonds with AT rich double stranded DNA. When introduced to cell cultures, it inhibits condensation of the heterochromatic region of the Y chromosome. The frequency of metaphases showing inhibition of heterochromatin condensation of the Y chromosome was found to be dependent on the treatment time and concentration of distamycin-A in the culture medium. When distamycin-A was added to a concentration of 100 micrograms/ml at the start of the culture (72 hours), the frequency of Y heterochromatin decondensation was found to be 48%, 30% and 6% in amniotic fluid, lymphocyte and fibroblast cultures respectively. The highest frequency of metaphases with decondensed Y heterochromatin were observed when distamycin-A treatment was carried out for the last 24 hours prior to harvest, the frequencies being 94%, 72% and 59% in amniotic fluid, lymphocyte and fibroblast cultures respectively. Increase in the concentration of distamycin-A from 25 micrograms/ml to 50 micrograms/ml during the last 24 hours of culture increased the incidence of metaphases with Y heterochromatin decondensation from 51% to 69% in amniotic fluid, 40 to 49% in lymphocyte and 29% to 31% in fibroblast cultures. Highest frequency of metaphases with Y heterochromatin decondensation were observed when the cultures were exposed to distamycin-A at a concentration of 100 micrograms/ml for the last 24 hours of culture.     

Description of the Anser anser goose karyotype

The karyotype of the Italian goose originating from Anser anser was characterised on the basis of R and C bands. Chromosomal preparations obtained from an in vitro culture of blood lymphocytes were stained with the RBG and CBG techniques. The RBG technique enabled the analysis of the structure of nine pairs of chromosomes whereas the CBG technique - fourteen pairs ofchromosomes from the total ofeighty goose chromosomes. The morphology and the R and C banding patterns were described. The size and arrangement of the blocks of constitutive chromatin were determined. Ideograms of R and C banded patterns were drawn. The morphological structure of the analysed chromosomes was evaluated.     

Variation in lysosomal enzyme activity during growth in culture of human fibroblasts and amniotic fluid cells

1. 1. The specific activity of the lysosomal hydrolases in cultured skin fibroblasts varies according to the phase of growth in culture.

2. 2. Diagnosis of heterozygous genotypes for lysosomal enzyme deficiency diseases is unreliable with cultured fibroblasts, at least partly because of the growth curve-associated variations in specific activity.

3. 3. Fluctuations in specific activity during the beginning of the growth curve in vitro can be avoided by initiating cultures with cells which are in the early log phase of growth.

4. 4. Primary amniotic fluid cell cultures show no relationship between length of time in culture and lysosomal enzyme specific activity.

5. 5. Secondary amniotic fluid cell cultures exhibit growth curve-related variations in lysosomal enzyme specific activity as they assume fibroblast-like growth kinetics.

6. 6. Prenatal and postnatal diagnosis on cultured amniotic fluid cells and fibroblasts requires the use of appropriate controls which are matched for stage of growth and length of time after the last trypsinization.

     

Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics

Summary DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with ⁶⁰Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.Dedicated to Professor Friedrich Vogel on the occasion of his 65th birthday     

A new and simple technique for chromosomal preparations from peripheral blood lymphocytes,amniotic cell cultures,skin fibroblasts,bone marrow and single cell clones when the yields from harvests are low

Summary A technique for chromosomal preparations from low yields harvests is presented using Poly-l-Lysine (300,000–400,000 MW). The procedure can be successfully used for peripheral blood lymphocytes, amniotic cell cultures, skin fibroblasts, direct preparations from bone marrow, cloned clusters and colonies from bone marrow grown on agar or methyl cellulose and eggs and blastocysts, with applications to other tissues. The technique is simple with minimal cell loss, and chromosomal preparations of good quality can be obtained which are amenable to all banding techniques.     

Triploidy in a fetus following amniocentesis referred for maternal serum screening test at second trimester

Amniocentesis was carried out at 17 weeks gestation in a 27-year-old woman, following an abnormal maternal serum screening (MSS) test. MSS test was carried out primarily to estimate the risk of trisomy for chromosome 21. The maternal serum markers used were alpha-fetoprotein (AFP), human chorionic gonadotrophin (hCG), and unconjugated estriol (uE3), together with maternal age. The fetus was identified as screen-positive for Edward's syndrome (trisomy 18), with low uE3, normal AFP and hCG levels. The calculated risk for trisomy 18 was more than 1:50. To identify any possible chromosomal abnormality, cytogenetic investigation was carried out on the amniotic fluid sample. The fetus's karyotype showed triploidy with 69, XXX chromosome complement in all the metaphase spreads obtained from three different cultures, using GTG banding technique. Upon termination of the fetus, gross abnormalities indicative of triploidy were present in the fetus.     

Chromosome lesions in amniotic fluid cell cultures

Summary The frequencies of chromosome lesions were determined on 3537 mitoses in samples of varying sizes from cultures of 25 amniotic fluid specimens taken from patients at cytogenetic risk. The average percentage values of aberrant cells, including and excluding gaps, were 12.5 and 4.9, respectively. The corresponding values for fibroblasts and peripheral blood lymphocytes from normal adult donors, calculated under the same laboratory conditions, were 5.0 (including gaps) and 2.4 (excluding gaps) and 2.4 (including gaps) and 1.0 (excluding gaps), respectively. The hypothesis of a correlation between the increased incidence of chromosome lesions and the occurrence of abnormal karyotypes in amniotic fluid cell cultures is discussed.     

Maternal cell contamination

Summary The problem of contamination of amniotic fluid cultures with maternal cells is described in 3 cases.