BUdR-Giemsa labeling and satellite association in human leukocytes

Summary Satellite associations were analysed in differentially stained human leukocyte chromosomes, obtained from four patients with Down's syndrome and four normal probands. A particular type of close association between two acrocentrics, showing a non-random arrangement of sister chromatids in a concordant dark-to-dark and light-to-light alignment, was found to be more common in patients with Down's syndrome compared with the normal controls. Apart from this particular type of association, sister chromatids are randomly arranged in satellite associations between two acrocentrics in both groups of probands. Considerable differences in the mean frequencies of satellite associations between first and second metaphases of the same individual were found in some probands of both groups of individuals. Since a high degree of inter-individual variability in the proliferative response of human leukocytes in culture is well established, the use of BUdR-Giemsa labeling for comparative analysis of satellite association frequencies is suggested.     

Chromatid associations in acrocentric chromosomes: evidence against nonrandomness.

Acrocentric chromosome associations from peripheral blood cultures of four normal individuals were examined after two replication cycles in bromodeoxyuridine (BrdU) using the FPG technique. Altogether, 167 out of 328 associations, or 51%, were concordant, having opposed chromatids similarly stained, and 49% discordant, thus indicating a random association of chromatids. None of the individual cultures revealed any significant departure from random chromatid association. Variation among individuals ranged from 38% to 58% concordance but repeat cultures did not indicate any consistent direction to the departure from 50%. Furthermore, neither the concentration of BrdU nor the type of association scored had any significant effect on the randomness of chromatid association. Thus, in contrast to another recent report, we found no evidence for a nonrandom alignment of chromatids in associated acrocentric chromosomes.     

NOR lateral asymmetry and its effect on satellite association in BrdU-labeled human lymphocyte cultures

Summary Second generation BrdU-labeled acrocentric chromosomes exhibit NOR lateral asymmetry (NLA) in metaphases that have been sequentially stained with silver and the Hoechst-Giemsa sister chromatid differential (SCD) technique. The NLA presumably results from suppression of NOR activity in the doubly-substituted chromatid. Examination of single chromatid (NOR) associations in pairs of acrocentrics reveals that light chromatids associate less frequently than dark chromatids and that the frequency distribution of dark and light alignment configurations can be explained by this differential tendency to associate. Thus, it appears that a hypothesis of non-random chromatid segregation as an explanation for non-random chromatid alignments in associating acrocentric chromosomes is unwarranted.This work is a joint project of The University of Texas M.D. Anderson Hospital and Tumor Institute and the John S. Dunn Research Foundation of Houston, Texas     

Analysis of exchanges in differentially stained meiotic chromosomes of Locusta migratoria after BrdU-substitution and FPG staining

In male mice the X and Y chromosomes are conjoined by a single near-terminal chiasma, but XY bivalents following incorporation of 5-bromodeoxyuridine (BrdU) and fluorescence plus Giemsa (FPG) staining show only one of the two expected configurations, which suggests a preferential involvement of certain non-sister chromatids in crossover formation. To test the possibility that nonrandom chromatid involvement is a general feature of near-terminal crossovers, we reexamined the apparently terminal associations in differentially stained autosomal bivalents of Locusta migratoria. The frequencies of the two configuration types were nearly equal, as would be expected if these terminal associations resulted from conventional near-terminal chiasmata showing the random involvement of non-sister chromatids that characterises interstitial chiasmata.     

Alignment of BrdU-substituted chromatids in satellite association in human metaphase cells

We have studied the orientation of BrdU-substituted chromatids in satellite associations in cells double-stained to reveal both the Ag/As nucleolar organizer regions and, simultaneously, sister chromatid differentiation. In those satellite associations with all four chromatids joined by Ag stain, and with the axes in a virtually straight line, we have observed an excess of concordant configurations. Where an association was considered single, dark chromatids were involved in significantly more associations than light chromatids. Within this group, the observed excess of concordant associations was not greatly different from the numbers observed in the straight, double-chromatid group of configurations. Whether the increased involvement of dark chromatids in satellite associations provides a complete explanation for the observed excess of concordant associations, or whether certain individuals show a specific tendency to form concordant associations, must await further data.     

Quantitative studies on the arrangment of human metaphase chromosomes

Summary The pattern of association of acrocentric chromosomes was examined in ten and five carriers of a 15/21 and a 13/14 Robertsonian translocation, respectively, and was compared with that of the same numbers of relatives with normal karyotypes. In the carriers of 15/21 translocation, the number of large associations (involving more than two acrocentrics) and the association frequencies for individual acrocentric chromosomes, were significantly higher than in the control group. The mean number of associations of the single homologs of the translocation chromosomes was much higher than that of the other acrocentrics. In the carriers of 13/14 translocations, only the association frequency for chromosome 13 was higher than in the normal relatives. The uninvolved chromosomes homologous to those involved in translocations showed an insignificant increase in associations in comparison with the other acrocentrics. These results suggest that some mechanism within the cells compensates for the effect of missing acrocentrics or of acrocentrics lacking NORs on the number of associations. The possible relations of this phenomenon to the activity of the nucleolus organizing regions are discussed.     

Characteristics of the cis- and trans-positions of chromatids of human acrocentric chromosomes in extreme old age

Cis- and trans-positions of chromatid associations of human acrocentric chromosomes were examined at extreme old age. Lymphocyte cultures were prepared by the usual method, from peripheral blood of 9 subjects aged 80-90 years (analysis of 179 metaphases) and 7 subjects aged 20-48 years (analysis of 124 metaphases). The functional difference between stalks of the sister chromatids was found. In the subjects at the age 80-90 years satellite stalks of chromatids-1 (in all DNA strands thymidine was substituted by 5-BrdU) of the D chromosome in cis-position are included into associations with lower frequency, as compared with the satellite stalks of chromatids-2 (thymidine+ is only substituted by 5-BrdU in a half DNA strands of the chromosome). This apparently reflects variability of regulation of functional activity of satellite stalks of sister chromatids.     

Random segregation of sister chromatids in developing chick retinal cells demonstrated in vivo using the fluorescence plus Giemsa technique

Experiments were designed to test whether nonrandom segregation of sister chromatids at mitosis has a role in the production of cell diversity during embryogenesis. Segregation was examined in vivo in retinal cells from embryonic chicks. Chromatids were labelled with bromouracil and stained by the fluorescence plus Giemsa technique. No evidence of nonrandom segregation was observed in a frequency distribution of pairs of bifilarly labelled sister chromatids at the third metaphase after the start of labelling. Nor was there evidence that chromatids from homologous chromosomes segregated nonrandomly. Nonrandom segregation is probably not a mechanism for cell diversification.     

The relationship between the frequency of participation of acrocentric chromosomes in formation of satellite associations and the duration of human lymphocyte cultivation with phytohemmaglutinin]

All acrocentrics were shown to take part in the formation of satellite association at the 52nd, 72nd and 90th hr of human lymphocytes culturing, in the first mitosis with equal frequency but having different associative capacity in further mitoses. It seems likely that a single nucleolus in the lymphocytes at initial stage of activation, the high frequency of satellite associations, and a random participation of acrocentrics in the associations in first mitosis are due to that all acrocentrics form association in small lymphocytes which are sensitive to PHA.     

Ag staining of the nucleolus organizer (NO) and its relationship to satellite association

Summary The frequency of involvement in satellite association and the frequency of selective staining of the secondary constrictions with silver solutions have been studied in five phenotypically normal individuals, all carriers of morphological variants of the nucleolus organizing region (NOR). The results show the preferential involvement of some morphological markers in satellite association, and also their preferential staining with Ag-I. It has also been shown that acrocentric chromosomes involved in satellite association are always stained by silver.     

Lateral asymmetry in human constitutive heterochromatin

Human lymphocytes were grown for one replication cycle in BrdU, stained with 33258 Hoechst, exposed to UV light and subsequently treated with 2 x SSC and stained with Giemsa. This technique differentially stains the constitutive heterochromatin of chromosomes 1, 9, 15, 16, and the Y. In the heterochromatin of chromosome 9 both sister chromatids stained darkly and symmetrically but in the other four chromosomes the heterochromatin showed lateral asymmetry, one chromatid being darkly stained while its sister chromatid was as pale or paler than the rest of the chromosome. The lateral asymmetry is presumed to reflect an underlying asymmetry in distribution of thymine between the two strands of the DNA duplex in the satellite DNA component of the chromosomes. In some number 1 chromosomes compound lateral asymmetry was seen; darkly staining material was present on both sister chromatids although at any given point lateral asymmetry was maintained so that if one chromatid stained darkly the corresponding point on the sister chromatid was very pale. The pattern of compound lateral asymmetry varied among the number 1 chromosomes studied but was constant for any one homologue from one individual. This technique reveals a previously unsuspected type of polymorphism within the constitutive heterochromatin of man.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> sister chromatids often show complete alignment or separation along a 1.2-Mb euchromatic region but no cohesion “hot spots”

Sister chromatid cohesion is a prerequisite for correct segregation and possibly other functions of replicated chromosomes. Except for yeast, no details are known about arrangement of cohesion sites along interphase chromosomes. Within nuclei of several higher plants, sister chromatids are frequently not aligned at various positions along chromosome arms. Therefore, we tested whether preferential alignment positions (“cohesion hot spots”) and constant extension of and distances between aligned sites occur in plants. Along a ~1.2-Mb contig from the bottom arm of chromosome 1, the sister chromatid positions of 13 individual BAC inserts were found to be aligned for ~67–77% of homologues in 4C Arabidopsis thaliana nuclei. The differences between the 13 BAC positions were not significant at the P < 0.01 level. This suggests variability of alignment positions between cells and indicates the absence of cohesion “hot spots”. Similar as for single BACs, FISH with the entire contig indicated complete alignment for ~69% and complete separation of sister chromatids for ~31% of homologues in 4C nuclei. Partial alignment or separation was barely detectable. When three BAC inserts from a 760-kb region were tested simultaneously, alignment or separation of only the central BAC occurred in 3.3% and 3.5% of replicated chromosomes, respectively. Thus, we assume that sister chromatids can be separated or aligned within a Mb range in differentiated cells. However, the minimum extension of aligned sites or distances between them may (in rare cases) fall below ~500 kb.     

Late separating D/D fusions in subjects with "balanced" translocation

The centromere separation sequence was determined in the mitoses of 5 subjects with "balanced" D/D translocations. Similarly to the acrocentrics, also the D/D fusions belonged to the chromosomes the sister chromatids of which separated last in the late metaphase stage.     

Comparison of acrocentric associations in male and female cells. Relationship to the active nucleolar organizers

Summary This work deals with a computer-aided study of associations between trypsin-banded acrocentrics in different male and female samples in a total of 900 cells. In female samples, acrocentrics 21 appeared as the most frequently involved in associations, followed by chromosome 13, whereas in male samples cell associations seemed more randomly distributed in the different samples.In addition, investigations in one female subject showed a very high correlation between the frequency with which an acrocentric was found in association and the presence and size of its active rDNA genes as shown by silver staining followed by staining with acridine orange.     

Random acrocentric bivalent associations in human pachytene spermatocytes. Molecular implications in the occurrence of Robertsonian translocations

Acrocentric bivalent associations were studied in 232 human male germ cells at pachytene in order to understand better the preferential involvement of chromosomes 13, 14, and 21 in Robertsonian translocations. The tendency of each acrocentric bivalent to associate with another was not correlated with NOR activity, as measured by silver staining. Good agreement was noticed between their ability to associate and the amount of satellite DNA in human acrocentric chromosomes. The distribution of two-by-two acrocentric bivalent associations was random. In order to reconcile this result with the nonrandom distribution of Robertsonian translocations, a molecular hypothesis is proposed. The model is based on homology of recombinational sites, interspersed at regular interval in satellite DNA, which could increase the probability of accidental unequal crossing-over between two specific acrocentric chromosomes.     

Evidence for the inheritance of silver-stained nucleolus organizer regions

Summary The inheritance of nucleolus organizer regions (NORs) was investigated by examining the degree of silver-staining in individual acrocentric chromosomes in two successive generations. The study was undertaken in six Down's syndrome children and their respective parents. Quinacrine fluorescent polymorphisms were used to identify individual acrocentrics and to determine which of the child's acrocentrics were informative as to parental homologue of origin. Of the 66 acrocentrics in the six children, 31 were informative. The correlation between the degree of silver-staining in the child's chromosomes and the respective parental chromosomes of origin was highly significant (P<0.001), with a correlation coefficient of 0.90. The results suggest that the degree of Ag-AS staining is characteristic for a particular chromosome and that this characteristic is an inherited property.     

Molecular Dissection of a Major Gene Effect on a Quantitative Trait: The Level of Alcohol Dehydrogenase Expression in Drosophila Melanogaster

A molecular mapping experiment shows that a major gene effect on a quantitative trait, the level of alcohol dehydrogenase expression in Drosophila melanogaster, is due to multiple polymorphisms within the Adh gene. These polymorphisms are located in an intron, the coding sequence, and the 3' untranslated region. Because of nonrandom associations among polymorphisms at different sites, the individual effects combine (in some cases epistatically) to produce ``superalleles' with large effect. These results have implications for the interpretation of major gene effects detected by quantitative trait locus mapping methods. They show that large effects due to a single locus may be due to multiple associated polymorphisms (or sequential fixations in isolated populations) rather than individual mutations of large effect.     

Frequency of satellite association of human chromosomes is correlated with amount of Ag-staining of the nucleolus organizer region.

Methaphase chromosomes from karyotypically normal adult humans (three males, six females) and one male with a 13p - chromosome were stained by quinacrine and then by the Ag-AS silver staining method to reveal nucleolus organizer regions (NORs). Each person had a characteristic number of Ag-stained chromosomes per cell, always fewer than 10. Determination of the mean Ag-size of each chromosome showed that each of the 10 individuals had a unique distribution of Ag-stain. Within each individual, there was some variation from cell to cell in the number of acrocentric chromosomes that were Ag-stained; this was not random, and the same chromosomes (those that had at most a small amount of Ag-stain) tended to be unstained in every cell. Satellite associations were scored on the same cells. Chromosomes that had no Ag-stain were involved in satellite association less than 20% as often as those that had some Ag-stain. Chromosomes that had a small amount of Ag-stain were involved in association about 50% as often as those that had a large amount of stain. Regression analysis of the 50 (of a total of 100) acrocentric chromosomes which could be individually identified by quinacrine markers showed that the frequency with which a chromosome was involved in satellite association was strongly correlated with the amount of Ag-stained material in the NOR.     

Maternal transmission ratio distortion at the mouse Om locus results from meiotic drive at the second meiotic division

We have observed maternal transmission ratio distortion (TRD) in favor of DDK alleles at the Ovum mutant (Om) locus on mouse chromosome 11 among the offspring of (C57BL/6 x DDK) F(1) females and C57BL/6 males. Although significant lethality occurs in this backcross ( approximately 50%), differences in the level of TRD found in recombinant vs. nonrecombinant chromosomes among offspring argue that TRD is due to nonrandom segregation of chromatids at the second meiotic division, i.e., true meiotic drive. We tested this hypothesis directly, by determining the centromere and Om genotypes of individual chromatids in zygote stage embryos. We found similar levels of TRD in favor of DDK alleles at Om in the female pronucleus and TRD in favor of C57BL/6 alleles at Om in the second polar body. In those embryos for which complete dyads have been reconstructed, TRD was present only in those inheriting heteromorphic dyads. These results demonstrate that meiotic drive occurs at MII and that preferential death of one genotypic class of embryo does not play a large role in the TRD.