Centromeres of mammalian chromosomes

The centromere is the major cis-acting genetic locus involved in chromosome segregation in mitosis and meiosis. The mammalian centromere is characterized by large amounts of tandemly repeated satellite DNA and by a number of specific centromere proteins, at least one of which has been shown to interact directly with centromeric satellite DNA sequences. Although direct functional assays of chromosome segregation are still lacking, the data are most consistent with a structural and possibly functional role for satellite DNA in the mammalian centromere.     

Homology of G-banded mammalian chromosomes

Linkage relationships of homologous loci and high resolution G-banding patterns of man, mouse, rat, chinese hamster, rabbit, cat, mink, pig, ox and sheep were used for identification of 11 evolutionary conservative autosomal regions. The distributions and inversions of these regions in the ancestor genomes of some phylums have been shown. For example, the regions homologous to human Ip region were detected in cat, mink and rabbit genomes. In the genomes of rodents studied the intraregion inversion was detected. In the ox and sheep genomes the distal end deletions were detected within these regions. In the pig genome these regions were represented solely by disruptions. We supposed that the rapid "catastrophic" chromosomal evolution took place during short time periods of some orders and families separation.     

Differential reactivity in mammalian chromosomes

Leucocyte cultures were treated with both ³H-thymidine and low temperature. Leucocyte cells were pulse labeled with ³H-thymidine for 15 to 20 minutes, and then placed in nonisotopic medium for 0, 1, 2, 3 and 4 hours respectively. Each culture was immediately treated with low temperature at 0–3° C for 24 hours. No metaphase chromosome were labeled at 0 and 1 hour after reincubation. Labeled metaphases were first observed after 2 hours of reincubation (3.9%); they increased after 3 hours (57%) and 4 hours of reincubation (39%). Labeled anaphases or telophases were also detectable in increasing proportions after 4 hours. Cell division proceeds very slowly through metaphase at low temperature. After labeling in the final 15 to 20 minutes of the S-period, one X-chromosome usually showed the late-replicating pattern. Label was found in the special segments of the X-chromosomes, X_E–a, X_L–a and X_L–b. Late-replicating regions in autosomes coincide more or less with the special segments. Differential reactivity in human chromosomes by low temperature was suggested to take place during the final part of G₂ after DNA synthesis.     

Comparative painting of mammalian chromosomes

Comparative chromosome painting has shown that synteny has been conserved for large segments of the genome in various placental mammals. Advances such as spectral karyotyping and multicolour ‘bar coding’ lend speed and precision to comparative molecular cytogenetics. Reciprocal chromosome painting and hybridisations with probes such as yeast artificial chromosomes, cosmids, and fibre fluorescence in situ hybridisation allow subchromosomal assignments of chromosome regions and can identify breakpoints of rearranged chromosomes. Advances in molecular cytogenetics can now be used to test the hypothesis that chromosome rearrangement breakpoints in human pathology and in evolution are correlated.     

Metaphase association of mammalian chromosomes

The association behavior of chromosomes bearing nucleolar organizer region (NOR) and (or) C-heterochromatin in metaphase plates was analyzed. Different species with an informative chromosomal localization of NOR and C-heterochromatin were evaluated. Several examples indicate that the well-known metaphase association is not due to NORs or NOR activity per se. Other mechanisms such as ectopic pairing are responsible for the association. These types of pairing seem to be enhanced by the chromatin-decondensing effect of nearby NOR activity.     

Origin and evolution of mammalian sex chromosomes

Mammals present an XX/XY system of chromosomal sex determination, males being the heterogametic sex. Comparative studies of the gene content of sex chromosomes from the major groups of mammals reveal that most Y genes have X-linked homologues and that X and Y share homologous pseudoautosomal regions. These observations, together with the presence of the two homologous regions (pseudoautosomal regions) at the tips of the sex chromosomes, suggest that these chromosomes began as an ordinary pair of homologous autosomes. Birds present a ZW/ZZ system of chromosomal sex determination where females are the heterogametic sex. In this case, avian sex chromosomes are derived from different pairs of autosomes than mammals. The evolutionary pathway from the autosomal homomorphic departure to the present-day heteromorphic sex chromosomes in mammals includes suppression of X-Y recombination, differentiation of the nascent non-recombining regions, and progressive autosomal addition and attrition of the sex chromosomes. Recent results indicate that the event marking the beginning of the differentiation between the extant X and Y chromosomes occurred about 300 million years ago.     

Highly conserved segments in mammalian chromosomes

Mammalian chromosomes from seven species for which gene maps exist were studied by high-resolution techniques to identify areas of conserved chromosome banding homology. High-resolution comparisons of human, chimpanzee, gorilla, orangutan, African green monkey, cat, and mouse chromosomes revealed regions of apparently conserved chromosomal banding, which may indicate the likely positions of conserved linkage in the phylogeny of mammals. This analysis indicates that many regions of subbanding homology may have remained intact during the evolution of mammals and reflects a high degree of chromosome conservation in diverse species.     

Differential spiralization along mammalian mitotic chromosomes

Morphology of chromosomes replicating in the presence of 5-bromodeoxyuridine was studied using long-term cultures of Chinese hamster cells (line Blld-ii-FAF28). The cytological effect of the analog administered in various concentrations, at different stages of the S period, and during one and two successive mitotic cycles was studied. — The main cytological manifestation of the BUdR action consisted in spiralization delay of certain chromosome regions. The degree of the delay was dependent on the time interval between the introduction of the agent and mitosis, as well as on the agent's concentration. With prolongation of the interval, the spiralization delay diminished and disappeared being therefore always observable only in late replicating chromosome regions. Increased concentration of BUdR (in the range of 25 to 400 g/ml) produced enhancement of the delay of chromosome spiralization. — After two successive reproduction cycles in the presence of BUdR, a great number of metaphases contained chromosomes the sister chromatids of which showed unequal spiralization delay. Autoradiography of ³H-BUdR distribution showed that the sister chromatid with a more pronounced underspiralization corresponds to the chromatid incorporating BUdR into both strands of the DNA molecule. — Mechanisms of the effect observed, as well as chemical influence on chromosome spiralization as a usefull tool of displaying linear chromosome differentiation, are discussed.     

Replication of DNA in mammalian chromosomes

A study of sedimentation and buoyant density of Okazaki fragments from mammalian chromosomes along with electron microscopic studies indicate that fragments from about 200 to 1200 nucleotides long may have RNA segments covalently attached. The fragments in some CsCl isopycnic gradients banded in two rather distinct bands. One band corresponds to the density of single-stranded DNA, but the other has a higher buoyant density which could be conferred by a segment of RNA up to 180 nucleotides or more in length. The RNA was not removed by denaturing conditions which separated DNA strands consisting of several thousand nucleotide pairs. When the material of higher buoyant density was spread for electron microscopy under conditions which would extend single-stranded DNA chains, but leave RNA in a coil or bush the chains with a higher buoyant density usually had a bush attached at one end. Under conditions that were thought to favor gap filling over chain elongation near growing forks, the DNA produced by pulse labeling with bromodeoxyuridine had a buoyant density which would indicate substitution to about 15 percent in one chain. If this substitution represents filling of gaps occupied by RNA before the pulse, the segments would be about 180 nucleotides in length assuming about 1,000 nucleotides between each segment.     

Arrangement and association of somatic chromosomes induced by chloramphenicol in barley

Chromosome number in the cells of the first division cycle in the root tip of Hordeum vulgare (2n=14) was apparently reduced from 2n to n by the chloramphenicol (CAP) treatments in early S, S late and/or early G ₂ stages. Morphological observations and measurements of relative DNA content per cell indicated that such reduction was due to tight alignment of chromosomes in pairs. —It was supposed that homologous chromosomes are close together in the interphase nucleus, and their successive association up to mitotic prometaphase was induced by the CAP treatment.     

Replication of DNA in mammalian chromosomes

DNA replication has been studied in cells (CHO) synchronized by mitotic selection from roller cultures. A study of the incorporation of ³H supplied as uridine indicates that cells cannot be blocked precisely at the beginning of the S phase, but DNA synthesis can be stopped in early S by treating with F-dU in G₁. After blockage potential initiation sites continue to increase at a linear rate for atleast 13 hours after division. Incorporation of ³H-thymidine begins at most of these sites within seconds after thymidine is supplied in the medium and incorporation continues at a linear rate for 20–24 minutes. There appears to be a pause after this interval before synthesis is resumed at about two times the initial rate. ³H-bromodeoxyuridine can be substituted for thymidine without affecting the kinetic pattern over a similar period. The increased rate is probably an increase in sites of chain growth rather than a change in rate of chain growth. A study of the labeled DNA segments by band sedimentation in a preformed NaClO₄ isokinetic gradient shows that two distinctly different sized segments can be released from the chromosomes by lysis at submelting conditions. One is the previously reported single chain segments averaging about one-half micron in length, but the other is a much larger segment (26S) which is native DNA with perhaps small regions of single chains presumably at the ends. Primarily single chain DNA is released after 1–2 minute pulse labeling, but after 2 minutes the larger segments (26S) contain most of the newly formed DNA except that attached to the chains of the major part of the template DNA which exhibits a discontinuous distribution, sedimenting far faster than either newly replicated segment. A consideration of the kinetics of formation of the 26S component indicates that is may contain the replicating fork. If this proves to be the correct interpretation the template chains would both have non-adjacent nicks preceeding the fork and also in a post-fork site at a mean distance of about 2 microns in both directions. The isolation of the growing points of DNA replication in chromosomes is now possible and the study of properties of the newly replicated regions should be greatly facilitated.     

Quantitative tritium autoradiography of mammalian chromosomes

The rates of tritiated thymidine accumulation in each of the chromosome types in Chinese hamster line Don and strain Don-C have been assayed by quantitative tritium autoradiography. The late-replicating nature of the X and Y chromosomes is readily apparent. Many chromosomes exhibit three separate steps of synthesis, with reduced rates of thymidine incorporation between 3 and 4 hours and again between 5 and 6 hours. The same three-step pattern can be seen in scintillation data from FUdR synchronized cells, with 40% of the DNA made in each of the first two steps and 20% in the final step.This research was supported in part by Grant GB-7248 from the National Science Foundation and by Grant E-286 from The American Cancer Society.     

Differential spiralization along mammalian mitotic chromosomes

The morphology of human metaphase chromosomes of peripheral blood lymphocytes taken from normal persons of both sexes and cultured at the final stages of the S-period in the presence of 5-bromodeoxyuridine (BUdR), or 5-bromodeoxycytidine (BCdR) was studied. It was observed that the chromosomes of the complement were capable of responsing to the treatment with analogs by the appearance of extended segments along their length. The pattern of segmentation was constant and specific for a given chromosome, serving as a basis for its identification, and appeared to be similar for both analogs. — Autoradiography of such chromosomes performed with ³H-thymidine (³H-TdR), ³H-deoxycytidine (³H-CdR), and ³H-BUdR showed that the extended chromosomal segments are late replicating. In accordance with this correlation, the most regular and distinctive segmentation was observed in chromosomes having large late replicating regions, such as Nos. 4, 6, 9, 13, 16, X, and Y. — A comparative analysis of the BUdR-induced differential spiralization pattern and banding pattern obtained with the G-staining technique was carried out. A good correspondence between the extended segments and Giemsa-positive bands was found. The data are discussed in relation to the mechanism of differential staining of metaphase chromosomes.