The chromosomes of CHO,an aneuploid Chinese hamster cell line: G-band,C-band,and autoradiographic analyses

Chinese hamster ovary cells (line CHO) have been used extensively for metabolic, genetic, and radiobiological studies with only a superficial appreciation for the degree of aneuploidy characteristic of the line. A thorough karyologic analysis of CHO chromosomes using autoradiographic replication patterns, as well as centromere band (C-band) and Giemsa band (G-band) analysis, is presented. Our results demonstrate that only 8 of the 21 CHO chromosomes are normal when compared with euploid Chinese hamster chromosomes. In the 13 altered chromosomes, we found evidence of translocations, deletions, and pericentric inversions. These altered chromosomes have been characterized with respect to both origin and destination of translocated material. With the exception of the X₂ chromosome, essentially all of the euploid chromatin is present in CHO cells. Autoradiographic replication patterns show that the normal sequence of chromosomal DNA synthesis is altered. Some sites which replicate late in euploid cells replicate early in CHO, and several late-replicating chromosomes in CHO cells replicate in early- or mid-S in euploid material. These studies may serve to elucidate the observed differences in mutagenic behavior between euploid fibroblasts and CHO cells.     

The distribution of sequences complementary to human satellite DNAs I,II and IV in the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus)

Human satellite DNAs I, II and IV were transcribed to yield radioactive complementary RNAs (cRNAs). These cRNAs were hybridised to metaphase chromosomes of man, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus). The results of this in situ hybridisation were analysed quantitatively and compared with accepted chromosome homologies based on Giemsa banding patterns. The cRNA to satellite II (cRNAII) did not hybridise to chimpanzee chromosomes, although its hybridisation to chromosomes of gorilla and orang utan yielded more autoradiograph grains than hybridisation to human chromosomes, and cRNAIV hybridised to many chromosomes of gorilla and chimpanzee but was almost entirely restricted to the Y chromosome in orang utan. Most sites of hybridisation were located on homologous chromosomes in all four species, but there were a number of sites which showed no correspondence between satellite DNA location and chromosome banding patterns, and others where a given chromosomal location hybridised with different cRNAs in each species. These results are in contrast to those found for many transcribed DNA sequences, where the same sequence is usually located at homologous chromosome sites in different species, and appear to cast doubt on many proposed models of satellite DNA function.     

Homologies in human and Macasa fuscata chromosomes revealed by in situ suppression hybridization with human chromosome specific DNA libraries

We established chromosomal homologies between all chromosomes of the human karyotype and that of an old world monkey (Macaca fuscata) by chromosomal in situ suppression (CISS) hybridization with human chromosome specific DNA libraries. Except for the human chromosome 2 library and limited cross-hybridization of X and Y chromosome libraries all human DNA libraries hybridized to single GTG-banded macaque chromosomes. Only three macaque chromosomes (2, 7, 13) were each hybridized by two separate human libraries (7 and 21, 14 and 15, 20 and 22 respectively). Thus, an unequivocally high degree of synteny between human and macaque chromosomes has been maintained for more than 20 million years. As previously suggested, both Papionini (macaques, baboons, mandrills and cercocebus monkeys, all of which have nearly identical karyotypes) and humans are chromosomally conservative. The results suggest, that CISS hybridization can be expected to become an indispensable tool in comparative chromosome and gene mapping and will help clarify chromosomal phylogenies with speed and accuracy.by E.R. Schmidt     

An ovarian chromosome map of Anopheles stephensi

A polytene chromosome map of Anopheles stephensi has been prepared from the ovarian nurse cells of adult females. Many homologous regions can be recognized in comparisons between the ovarian and salivary gland chromosome maps but band for band homologies are not readily evident. The preparation of polytene chromosomes from ovarian nurse cells is easier than from the larval salivary glands and the results more consistent.     

DNA replication asynchrony between the paternal and maternal alleles of imprinted genes does not straddle the R/G transition

Imprinted autosomal loci apparently reside in very large chromosomal domains that exhibit asynchrony in replication of homologous alleles during the DNA synthesis phase. Replication asynchrony can be cytogenetically visualized by a replication-banding discordance between homologous bands of a given pair of chromosomal homologs. The replication time of a chromosomal band at high resolution can be determined by blocking DNA synthesis at the R/G-band transition and using replication banding. The R/G transition reflects the transition from early (R-) to late (G- and C-) band DNA replication. We studied discordance between two groups of homologous chromosomal bands: (a) four bands, 6q26–27, 11p13, 11p15.5 and 15q11.2–12, each containing at least one imprinted gene; and (b) nine bands containing no known imprinted genes. Fifty pairs of chromosomes were analyzed at high resolution after R/G transition blocking and late 5-bromo-2′-deoxyuridine incorporation. The rate of discordance was the same for bands containing imprinted genes and for control bands. Both homologous bands of a pair replicate either before or after the R/G transition and do not straddle the R/G transition. Repression associated with imprinting does not appear to involve late replication at the band level of resolution. Tissue-specific inactivation is associated with DNA methylation and late replication, whereas allele-specific inactivation is associated with DNA methylation but not with delayed or late replication. Received: 7 May 1996; in revised form: 27 January 1997 / Accepted: 31 July 1997     

High-resolution analysis of DNA replication domain organization across an R/G-band boundary.

Establishing how mammalian chromosome replication is regulated and how groups of replication origins are organized into replication bands will significantly increase our understanding of chromosome organization. Replication time bands in mammalian chromosomes show overall congruency with structural R- and G-banding patterns as revealed by different chromosome banding techniques. Thus, chromosome bands reflect variations in the longitudinal structure and function of the chromosome, but little is known about the structural basis of the metaphase chromosome banding pattern. At the microscopic level, both structural R and G bands and replication bands occupy discrete domains along chromosomes, suggesting separation by distinct boundaries. The purpose of this study was to determine replication timing differences encompassing a boundary between differentially replicating chromosomal bands. Using competitive PCR on replicated DNA from flow-sorted cell cycle fractions, we have analyzed the replication timing of markers spanning roughly 5 Mb of human chromosome 13q14.3/q21.1. This is only the second report of high-resolution analysis of replication timing differences across an R/G-band boundary. In contrast to previous work, however, we find that band boundaries are defined by a gradient in replication timing rather than by a sharp boundary separating R and G bands into functionally distinct chromatin compartments. These findings indicate that topographical band boundaries are not defined by specific sequences or structures.     

Analysis of DNA replication during S-phase by means of dynamic chromosome banding at high resolution

The characteristic patterns of dynamic banding (replication banding) were analysed. Extremely high resolution (850 to 1,250 bands per genome) G- and R-band patterns were obtained after 5-bromo-2-deoxyuridine (BrdUrd) incorporation either during the early or the late S-phase. We synchronized human lymphocytes with high concentrations of thymidine or BrdUrd as blocking agents, followed by low concentrations of BrdUrd or thymidine respectively as releasing agents, and obtained R- or G-band patterns respectively. The dynamic R-and G-band patterns were complementary for all chromosomes, even for the late-replicating X chromosome. There was no overlapping and every part of each chromosome was positively stained by one of the two banding procedures. The complementarity of the two patterns shows that both high thymidine and high BrdUrd concentrations blocked S-phase progression near the R-band to G-band replication transition in the middle of S-phase. Some bands of the inactive X chromosome replicate before this transition concurrently with R-band replication. The 48 different telomeric regions could be classified into 5 distinct morphotypes based upon the distribution of early and late-replicating DNA in each telomeric region. The dynamic band patterns are particularly useful for the study of the structural and physiological organization of chromosomes at high resolution and should prove invaluable for assessing the replication behavior of rearranged chromosomes.     

The human X-linked steroid sulfatase gene and a Y-encoded pseudogene: evidence for an inversion of the Y chromosome during primate evolution

The mammalian X and Y chromosomes are thought to have evolved from a common, nearly homologous chromosome pair. Although there is little sequence similarity between the mouse or the human X and Y, there are several regions in which moderate to extensive sequence homologies have been found, including, but not limited to, the so-called pseudoautosomal segment, in which X-Y pairing and recombination take place. The steroid sulfatase gene is in the pseudoautosomal region of the mouse, but not in man. We have cloned and characterized the human STS X-encoded locus and a pseudogene that is present on the long arm of the Y chromosome. Our data in humans and other primates suggest that there has been a pericentric inversion of the Y chromosome during primate evolution that has disrupted the former pseudoautosomal arrangement of these genes. These results provide additional insight into the evolution of the sex chromosomes and into the nature of this interesting portion of the human genome.     

Extensive sequence homologies between Y and other human chromosomes

Twenty-six human Y-chromosome-derived DNA sequences, free of repetitive material, were used to probe male and female genomic blots. We present data from a detailed analysis and chromosomal location of the bands detected by such probes, which demonstrate extensive DNA sequence homology between the mammalian sex chromosomes and autosomes. Under stringent conditions, nine Y-derived probes reacted exclusively with the Y chromosome, 12 probes detected homologous sequences present on both the Y and the X, four probes detected homologies between Y and autosome(s) without any X counterpart and, finally, one probe hybridized to homologous sequences on Y, X and autosome(s). These data are consistent with the hypothesis of a common evolutionary origin for the mammalian sex chromosomes and reveal structural similarities between Y-located and autosomal non-repetitive sequences.     

Comparative analysis of the cattle and human genomes: detection of ZOO-FISH and gene mapping-based chromosomal homologies

Comparative chromosome painting with individual human chromosome-specific libraries (CSLs) on cattle metaphase chromosomes delineated 46 homologous chromosomal segments between the two species. Continuous arrangement of these segments on individual cattle chromosomes demonstrates a nearly complete coverage of the bovine karyotype and shows physical boundaries of bovine chromosomal segments homologous to individual human chromosomes. Alignment of the available comparative gene mapping data with the homologous segments strongly supports the detected gross homologies between the karyotypes of the two species. In addition to cattle, four human CSLs were hybridized to sheep metaphase chromosomes also, to further verify the known karyotype homology within the Bovidae. Besides its application to karyotype evolution research, the comparative knowledge provides for rapid expansion of the much needed Type I locus-based bovine gene map. Received: 9 September 1995 / Accepted: 4 December 1995     

Conservation of whole arms during chromosomal divergence of phyllotine rodents

The assumption of simple fusion in a group showing a constant number of chromosome arms was tested by comparison of the G-band patterns of chromosomes of three Phyllotis species. The karyotypes, each of which has 40 chromosome arms, have a 2n of 38, 38, and 40 and are made up of mostly metacentric chromosomes. Operational concepts describing the amount of matching in G-band patterns are proposed, separating chromosomes or arms into those with total correspondence, partial correspondence, or unique cases. Seven chromosomes and 21 arms out of the total were identical in the three species, denoting a conservation of whole-arm band sequences in this group. A greater number of identical arms than of identical chromosomes were observed, giving some support to the simple fusion hypothesis. An unexpected chromosomal divergence was detected, including chromosomal variation in the C-banded sex chromosomes.     

KOPS: DNA motifs that control E. coli chromosome segregation by orienting the FtsK translocase

Bacterial chromosomes are organized in replichores of opposite sequence polarity. This conserved feature suggests a role in chromosome dynamics. Indeed, sequence polarity controls resolution of chromosome dimers in Escherichia coli. Chromosome dimers form by homologous recombination between sister chromosomes. They are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific chromosomal site, dif, and a DNA translocase, FtsK, which is anchored at the division septum and sorts chromosomal DNA to daughter cells. Evidences suggest that DNA motifs oriented from the replication origin towards dif provide FtsK with the necessary information to faithfully distribute chromosomal DNA to either side of the septum, thereby bringing the dif sites together at the end of this process. However, the nature of the DNA motifs acting as FtsK orienting polar sequences (KOPS) was unknown. Using genetics, bioinformatics and biochemistry, we have identified a family of DNA motifs in the E. coli chromosome with KOPS activity.     

Chromosome banding and gene localizations support extensive conservation of chromosome structure between cattle and sheep.

By using three gene probes, one derived from the porcine major histocompatibility complex (MHC) and two from bovine cytokeratin genes, type I (KRTA) and type II (KRTB), the hypothesis of conservation of genome structure in two members of the family Bovidae was examined. Gene mapping data revealed the MHC to be in chromosome region 23q15----q23 in cattle (BOLA) and 20q15----q23 in sheep (OLA). KRTA was localized to chromosome region 19q25----q29 in cattle and 11q25----q29 in sheep and KRTB to 5q14----q22 in cattle and 3q14----q22 in sheep. The banding patterns of the chromosome arms to which the loci were assigned were identical in both species. Moreover, the resemblances of GTG- or QFQ-banding patterns between the cattle and sheep karyotypes illustrated further chromosome homologies. These studies, based on gene mapping comparisons and comparative cytogenetics, document that within bovid chromosomes, homology of banding patterns corresponds to a homologous genetic structure. Hence, we propose that gene assignments on identified chromosomal segments in one species of the Bovidae can be extrapolated, in general, to other bovid species based on the banding homologies presented here.     

The late replication banding patterns of chromosomes are highly conserved in the genera Rana,Hyla, and Bufo (Amphibia: Anura)

Late replication banding and C-banding analyses were performed on the metaphase chromosomes of six species and one subspecies of Palearctic water frogs, genus Rana. Although C-banding patterns showed interspecific or intersubspecific variation, late replication banding patterns of all 13 chromosome pairs of these species were homologous. Minor differences of banding patterns were observed only in chromosomes 2, 7 and 13. Close comparison of the late replication banding patterns with those of three non-water frog species of Rana, and one each of Hyla and Bufo, provided important information on interspecific and intergeneric variability. In the Rana species, the banding patterns of all 13 pairs were homologous except for those some regions of 8 pairs. In one species each of Hyla and Bufo that was examined, the six large chromosome pairs (Nos. 1-6) showed banding homologies. Furthermore, among the Rana, Hyla and Bufo species the four large chromosome pairs (Nos. 1-3, 5 of Rana and Hyla, and Nos. 1, 3–5 of Bufo) shared banding homologies. These results show that the large chromosomes have been highly conserved in the evolutionary history of the three genera.     

Molecular and classical cytogenetic analyses demonstrate an apomorphic reciprocal chromosomal translocation in Gorilla gorilla.

The existence of an apomorphic reciprocal chromosomal translocation in the gorilla lineage has been asserted or denied by various cytogeneticists. We employed a new molecular cytogenetic strategy (chromosomal in situ suppression hybridization) combined with high-resolution banding, replication sequence analysis, and fluorochrome staining to demonstrate that a reciprocal translocation between ancestral chromosomes homologous to human chromosome 5 and 17 has indeed occurred.     

Replication timing in a single human chromosome 11 transferred into the Chinese hamster ovary (CHO) cell line

DNA replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict and often tissue-specific temporal program. However, the genetic program that controls activation of replication origins has still not been fully elucidated in mammalian cells. Previously, we measured replication timing at the sequence level along human chromosomes 11q and 21q. In the present study, we sought to obtain a greater understanding of the relationship between replication timing programs and human chromosomes by analysis of the timing of replication of a single human chromosome 11 that had been transferred into the Chinese hamster ovary (CHO) cell line by chromosome engineering. Timing of replication was compared for three 11q chromosomal regions in the transformed CHO cell line (CHO(h11)) and the original human fibroblast cell line, namely, the R/G-band boundary at 11q13.5/q14.1, the centromere and the distal telomere. We found that the pattern of replication timing in and around the R/G band boundary at 11q13.5/q14.1 was similar in CHO(h11) cells and fibroblasts. The 11q centromeric region, which replicates late in human fibroblasts, replicated in the second half of S phase in CHO(h11) cells. By contrast, however, the telomeric region at 11q25, which is late replicating in fibroblasts (and in several other human cell lines), replicated in the first half of S phase or in very early S phase in CHO(h11) cells. Our observations suggest that the replication timing programs of the R/G-band boundary and the centromeric region of human chromosome 11q are maintained in CHO(h11) cells, whereas that for the telomeric region is altered. The replication timing program of telomeric regions on human chromosomes might be regulated by specific mechanisms that differ from those for other chromosomal regions.     

Mapping G-bands on human prophase chromosomes.

OHNUKI's method for demonstrating coils in human metaphase chromosomes also reveals a fine G-band pattern on prophase chromosomes of sufficient clarity to justify an attempt at mapping. Maps are provided for each chromosome to show the maximum number of prophase bands observed, and an intermediate stage in chromosome contraction, tracing the pathways of apparent band fusion as the cell progresses to metaphase, is presented. The prophase bands on many chromosomes tend to occur in distinct groups, the members of which ultimately merge to give the dark G-bands of metaphase chromosomes. Every G-band of the standard metaphase chromosomes. Every G-band of the standard metaphase pattern is compounded from two or more prophase bands. In at least contracted prophase chromosomes examined, some bands are seen which have no obvious metaphase counterpart. There are marked similarities between banded prophases and the chromoomere pattern seen at meiotic prophase. However, since chromosome contraction is a dynamic process, agreement between maps will be expected only for corresponding degrees of chromosome contraction.     

The location of DNA homologous to human satellite III DNA in the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus)

Radioactive RNA with sequences complementary to human DNA satellite III was hybridised in situ to metaphase chromosomes of the chimpanzee (Pan troglodytes), the gorilla (Gorilla gorilla) and the orangutan (Pongo pygmaeus). A quantitative analysis of the radioactivity, and hence of the chromosomal distribution of human DNA satellite III equivalent sequences in the great apes, was undertaken, and the results compared with interspecies chromosome homologies based upon Giemsa banding patterns. In some instances DNA with sequence homology to human satellite III is present on the equivalent (homologous) chromosomes in identical positions in two or more species although quantitative differences are observed. In other cases there appears to be no correspondence between satellite DNA location and chromosome homology determined by banding patterns. These results differ from those found for most transcribed DNA sequences where the same sequence is located on homologous chromosomes in each species.     

Chromosome mapping of Miller-Diecker,Smith-Magenis and RARA loci in non-human primates: implications in the evolution of human chromosome 17

Molecular cytogenetics allows to verify chromosomal homologies previously hypothesised on the base of banding pattern comparison in different species. So far only the chromosome painting technique has been extensively used in studies of chromosomal evolution. This technique allows to detect only interchromosomal rearrangements. Human and Great Apes chromosomes basically differ by intrachromosomal rearrangements, in particular inversions; with chromosome painting it has just been possible to confirm the origin by fusion of human chromosome 2 and a reciprocal translocation in Gorilla, involving the homologous of chromosome 5 and 17. In order to verify intrachromosomal rearrangements in human chromosomal evolution, chromosome mapping of human loci in non-human primates is a useful approach. We mapped Miller-Diecker, Smith-Magenis and RARA loci localised on human chromosome 17, in Gorilla gorilla, Pongo pygmaeus, Macaca fascicularis and Cercopithecus aethiops. On the base of the obtained results it was possible to verify chromosomal rearrangements previously identified by banding, to achieve new informations about the controversial evolution of human chromosome 17, and to detect the occurrence of a paracentric inversion in the homologous in Cercopithecus aethiops.     

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.