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Gene mapping in Mus musculus by interspecific cell hybridization: assignment of the genes for tripeptidase-1 to chromosome 10, dipeptidase-2 to chromosome 18, acid phosphatase-1 to chromosome 12, and adenylate kinase-1 to chromosome 2.

Chinese hamster X mouse somatic cell hybrids segregating mouse chromosomes were examined for their mouse chromosome content using trypsin-Giemsa (GTG) banding and Hoechst 33258 staining techniques. Simultaneously, they were scored for the presence of 24 mouse enzymes. The results confirm the assignments of 11 genes previously mapped by sexual genetics: Dip-1 and Id-1 to chromosome 1; Pgm-2 and Pgd to 4; Pgm-1 to 5; Gpi-1 to 7; Gr-1 to 8; Mpi-1 and Mod-1 to 9; Np-1 and Es-10 to 14. They also confirm chromosomally the assignments of 3 genes that were made by other somatic cell genetic studies: Aprt to 8; Hprt and alpha-gal to the X chromosome. But most importantly, four enzyme loci are assigned to four chromosomes that until now were not known to carry a biochemical marker which is expressed in cultured cells: Trip-1 to 10; Dip-2 to 18; Acp-1 to 12; and Ak-1 to 2. Cytogenetic examination of clones showing discordant segregation of HPRT and A-GAL, suggested the assignment of alpha-gal to region XE leads to XF of the mouse X chromosome. The cytologic studies provide a comparison between data from sexual genetics and somatic cell hybrids and validate hybrid cell techniques. They provide evidence of the reliability of scoring chromosomes by GTG and Hoechst staining and stress the importance of identifying clones with multiple chromosome rearrangements. Striking examples of norandom segregation of mouse chromosomes were observed in these hybrids with preferential retention of 15 and segregation of 11 and the Y chromosome.

Acid Phosphatase

Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers.

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers. Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure of the spindle or the positions of the nonmanipulated chromosomes. From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery. In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle; however, the chromosome resists displacement away from the spindle pole. When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole. The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate. No evidence was found for mechanical attachments between separating half-bivalents in anaphase. Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force. Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy.

Animals

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle.

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement.

Animals

A pericentric inversion of chromosome 9 and a rearrangement involving chromosomes 9 and 10, observed in two generations. Clinical description of chromosome 9 (p12-p21) deletion syndrome.

The clinical picture associated with a deletion of a central part of the short arm of chromosome no. 9 is described in two siblings. The clinical signs differ from those described in deletion of the terminal part of the short arm. Pericentric inversion of chromosome no. 9, combined with a rearrangement involving chromosomes 9 and 10, was found in the mother and the maternal grandmother of the propositus.

Abnormalities, Multiple

Gene localization by chromosome fractionation: globin genes are on at least two chromosomes and three estrogen-inducible genes are on three chromosomes.

Chicken metaphase chromosomes were partially purified by rate zonal centrifugation, and DNA was prepared from each of the fractions of the sucrose gradient. The DNA was digested with various restriction enzymes and subjected to electrophoresis in agarose gels. The DNA was transferred to nitrocellulose filters (as described by Southern), and the filters were hybridized with cDNA probes. Four globin genes alpha A, alpha D, beta, and rho or epsilon are located on at least two chromosomes, and three of the estrogen-inducible genes of the hen oviduct--ovalbumin, ovomucoid, and transferrin--are on three different chromosomes. These experiments also confirm our earlier assignment of the endogenous viral sequence related to Rous-associated virus-0 to a separate (and larger) chromosome than the cellular sequence related to the transforming gene of avian sarcoma virus (cellular sarc), although it now appears that cellular sarc is on a small macrochromosome, rather than on a microchromosome.

Animals

Mapping of human chromosomal regions related to neoplasia: evidence from chromosomes 1 and 17.

In clonal aberrations leading to an excess or partial excess of chromosome 1, trisomy for bands 1q25-1q32 was noted in the myeloid cells from all of 34 patients who had various disorders such as acute leukemia, polycythemia vera, and myelofibrosis. This was not the result of a particularly fragile site in that region of the chromosome because the break points in reciprocal translocations that involve it occurred almost exclusively in the short arm. Two consistent rearrangements that have been observed in chromosome 17 produced either duplication of the entire long arm or a translocation of the distal portion of the long arm to chromosome 15. The nonrandom chromosomal changes found in hematologic disorders can now be correlated with the gene loci on these chromosomes or chromosomal segments. Seventy-five genes related to various metabolic enzymes have been mapped; it may be significant that chromosomes carrying gene loci related to nucleic acid metabolism are more frequently involved in hematologic disorders (and other malignancies as well) than are gene loci related to intermediary or carbohydrate metabolism. Furthermore, the known virus-human chromosome associations are closely correlated with the chromosomes affected in hematologic disorders. If one of the effects of carcinogens (including viruses) is to activate genes that regulate host cell DNA synthesis, and if translocations or duplications of specific chromosomal segments produce the same effect, then either of these mechanisms might provide the affected cell with a proliferative advantage.

Chromosome Aberrations

Preferential derivation of abnormal human G-group-like chromosomes from chromosome 15.

The marked binding of antibodies specific for 5-methylcytidine to the short arm of chromosome 15 distinguishes this chromosome from the other human acrocentrics. This method has been used to study over 60 individuals including 12 who did not have Down's syndrome, but who did have an extra G-group sized acrocentric chromosome. In six cases the extra chromosome did not show intensive binding of anti-5-methylcytidine. In the other six cases, the extra chromosome contained a 5-methylcytidine rich band at each end indicating that both ends were derived from chromosome 15 and contained centromeric heterochromatin normally present on the short arm of chromosome 15. The duplication of short arm material in the abnormal chromosomes was confirmed in all cases by quinacrine staining, nucleolar organizer (Ag-AS) staining or C-banding. In three cases, the abnormal chromosome appeared to arise from two different chromosomes 15. Several possible mechanisms for the production of the abnormal chromosome are discussed. The individuals with this abnormal chromosome all showed some degree of mental retardation, but few common physical findings.

Adult

Assignment of human beta-, gamma-, and delta-globin genes to the short arm of chromosome 11 by chromosome sorting and DNA restriction enzyme analysis.

Normal human metaphase chromosomes isolated from fibroblasts were resolved into 14 peaks based on total Hoechst 33258 fluorescence and sorted with the fluorescence-activated cell sorter. The chromosomal DNA was extracted and characterized by EcoRI analysis. As expected, analysis of the peak containing chromosomes 16 and 18 detected the alpha-globin genes and of the peak containing chromosomes 9, 10, 11, and 12 detected the beta-, gamma-, and delta-globin genes. Translocations were then used to localize further the beta-, gamma-, and delta-globin genes. The first translocation t(11;22)(q25;q11), which moved nearly all of chromosome 11 to a different peak, confirmed that the beta-, gamma-, and delta-globin genes are on this chromosome. The second, t(4;11)(q25;q13), which moved the distal portion of the long arm of chromosome 11 to a new peak, showed that the genes are not in this segment. The third, t(X;11)(q11;p13), moved the distal region of the short arm of chromosome 11 to a peak which now contained the beta-, gamma-, and delta-globin genes. Therefore, the beta-, gamma-, and delta-globin genes residue on the distal portion of the chromosome 11 short arm including bands p13, p14, and p15. This sorting method may be used generally to assign other genes to chromosomal segments of the entire chromosome complement.

Cell Fractionation

The role of chromosomal proteins in the C-banding of Allium cepa chromosomes.

When chromosomes of Allium cepa are subjected to a C-banding procedure (incubation in saturated barium hydroxide followed by phosphate buffer at 60 degrees C for 1 h) and then treated with Giemsa stain, bands appear at the telomeres of all chromosomes. Microspectrophotometric measurements of Feulgen-DNA content, demonstrated that the C-banding procedure extracted DNA from the nuclei. Staining of banded chromosomes with several DNA-specific stains showed that this loss was differential, with the band DNA exhibiting more resistance to extraction than that of the rest of the chromosome. The C-banding procedure did not extract chromosomal proteins, however, and no difference in mass per unit length could be detected by Nomarski optics between band and interband regions. Several experiments demonstrated that chromosomal proteins play a significant role in C-banding. First, treatment of chromosomes with pronase before C-banding resulted in the elimination of differential staining with Giemsa. Furthermore, in preparations where the DNA was completely hydrolysed with hot TCA, the remaining chromosomal proteins were found to exhibit a differential affinity for Giemsa stain. Amido black staining demonstrated that total chromosomal protein was uniformly distributed after the hot TCA digestion, but the proteins localized in the telomeres had a greater affinity for the Giemsa stain than the bulk of the chromosomal proteins. When the TCA-digested chromosomes were subjected to the C-banding procedure before staining, the differential affinity of the telomeres for the Giemsa stain was lost. Thus, C-banding appears to be the result of a complex interaction between protein and DNA in which the greater resistance to extraction of the band DNA is necessary to stabilize and preserve chromatin protein which exhibits a differential affinity for Giemsa stain.

Chromosome Banding

Chromosomal localization of human beta globin gene on human chromosome 11 in somatic cell hybrids.

We have successfully used a DNA.cDNA molecular hybridization assay to directly determine the presence or absence of human beta globin gene sequences in 20 human-mouse somatic cell hybrids, each of which contained a different subset of human chromosomes. The assay is specific for the individual human globin genes and will detect the presence of a globin gene if the relevant chromosome is present in only 10% of the cells of a hybrid population. The content of human chromosomes in each hybrid clone was characterized by Giemsa 11 staining, Giemsa trypsin-Hoechst 33258 staining, and by the use of 22 independent isozyme markers for 17 different human chromosomes. All human chromosomes were present in one or more cell lines devoid of the human beta globin gene except for 6, 8, 9, 11, and 13. Among these latter chromosomes, only chromosome 11 was present in the six hybrid clones that contained the human beta globin gene. In fact, chromosome 11 was the only human chromosome that was present in all of the six hybrid clones found to be positive for the human beta globin gene. Two sister clones, 157-BNPT-1 and 157-BNPT-4, had similar subsets of human chromosomes except that 11 was present only in 157-BNPT-4. 157-BNPT-4 contained the human beta globin gene while 157-BNPT-1 did not. DNA from three hybrid lines was also annealed to purified human gamma globin cDNA; two lines positive for human beta globin gene sequences also contained human gamma globin gene sequences while one line was negative for both beta and gamma gene sequences. On the basis of these results, the human beta and gamma globin genes have been assigned to human chromosome 11.

Chromosomes, Human, 6-12 and X

[Variability of the C-segment sizes of chromosomes 1, 9, 16 and Y in the human chromosome set].

The investigation of chromosome polymorphism by quantitative methods is a rather hard task. The manual method for measuring C-segments of chromosomes 1, 9, 16 and Y in man is suggested, which is not difficult, being reasonably precise for the population research. Metaphases of the average level of chromosome condensation were taken for analysis. Only the C-segments were measured without measuring chromosomes. The negative chromosome image was 4000-fold magnified, compared to the chromosome natural size, and the boundaries of C-segments of each chromosome were five-fold dotted on a sheet of paper specially printed for this purpose. C-segments were measured by magnifying glass with 0.025 mcm scale unit. For every individuum, C-segments were measured in 5-7 cells only. The data are presented on the estimation of measurement errors and on individual (intercellular) and population (interindividual) variations of C-segments of chromosomes.

Adolescent

Three chromosomes' (7;9;22) rearrangement and the origin of the Philadelphia chromosome.

A woman with chronic myelocytic leukemia had the Philadelphia chromosome and a complex four-break--three-chromosome rearrangement. The q32 leads to q34 portion of chromosome 9 is translocated to band q22 of chromosome 7, and at the end of this segment is attached the deleted q11 leads to qter portion of chromosome 22. A review of 12 cases of the Philadelphia chromosome originating by the rearrangement of three or more chromosomes reveals that chromosomes 9 and 22 are always involved, while the third chromosome is a different one in each case. We discuss the hypothesis that the 22q segment is always specifically attached to band 9q34 wherever this portion of 9q is transposed.

Aged

Chromosomal basis of dosage compensation in Drosophila. IX. Cellular autonomy of the faster replication of the X chromosome in haplo-X cells of Drosophila melanogaster and synchronous initiation.

[(3)H]Thymidine labeling patterns have been examined in gynandric mosaic salivary glands of drosophila melanogaster. The Ring-X stock, R(1) w(ve)/In(1)dl 49, l (1) J1 y w lz(s), was used for this purpose. 365 labeled XX2A and 40 labeled XO2A nuclei were obtained from a total of 624 nuclei in nine pairs of mosaic salivary glands. It was observed that in all but those nuclei which had DD, 1C, and 2C patterns, the X chromosome of the XO2A nuclei always had fewer sites labeled than the X chromosomes of the XX2A nuclei, for a given pattern of the autosomes in either sex. Such asynchronous labeling of the X chromosome in the XO2A (male) nuclei was observed regardless of the proportion of the XO2A cells (2.0-73.7 percent), in the mosaic glands. Moreover, while the frequency of [(3)H]thymidine labeling for all of the 39 replicating units except the two late replicating sites (3C and 11A) in the X chromosome of the XO2A nuclei, was consistently lower than in the X chromosome of the XX2A nuclei, the mean number of grains on the X chromosome was relatively (to autosomes) similar in both XX2A and XO2A cells. The results, therefore, suggest that, as in XY2A larval glands, the X chromosome in the XO2A cells also completes the replication earlier than autosomes and that the XO2A nuclei show cellular autonomy with respect to the early replication of the X chromosome, like its counterpart, RNA transcription. Absence of the asynchrony during the initial phase (DD-2C) further completes the replication earlier but that the rate of replication of its DNA is possibly faster, and (b) that there might be a common regulation with respect to the initiation of replication of different chromosomes in a genome.

Animals

Chromosome banding pattern conservatism in birds and nonhomology of chromosome banding patterns between birds, turtles, snakes and amphibians.

The G-banded karyotypes of 4 species of birds representing the orders Galliformes, Columbiformes and Musophagiformes were compared. Banding pattern homology between orders was limited t 5o 5 major chromosome arms and the Z chromosome. Even in these major chromosome arms pericentric and paracentric inversions produced alteration of the banding pattern sequences. Addition of constitutive heterochromatin was responsible for changes in banding patterns in the Z chromosome. The chromosome banding patterns of an emydid turtle, Terrepene carolina, 5 species of boid snakes of the genera Liasis, Acrantophis, and Sanzinia and the African clawed-frog. Xenopus muelleri, were also compared to the bird chromosome banding patterns. No homology was observed between any of these major groups: bird, snake, turtle, amphibian. However, intergroup homology was apparent. - The data obtained do not support reports of broad interordinal direct homology of the macrochromosomes of birds and refutes the idea of a primitive bird karyotype with 3 pairs of "Agroup' chromosomes and 3 pairs of "B group' chromosomes. - The major mechanisms responsible for chromosome evolution in birds appear to be centric and tandem fusions, paracentric and pericentric inversions, and addition or deletion of heterochromatin.

Animals

Germline-restricted chromosome of songbirds has different centromere compared to regular chromosomes.

Centromeres are an important part of chromosomes which direct chromosome segregation during cell division. Their modifications can therefore explain the unusual mitotic and meiotic behaviour of certain chromosomes, such as the germline-restricted chromosome (GRC) of songbirds. This chromosome is eliminated from somatic cells during early embryogenesis and later also from male germ cells during spermatogenesis. Although the mechanism of elimination is not yet known, it is possible that it involves a modification of the centromeric sequence on the GRC, resulting in problems with the attachment of this chromosome to the mitotic or meiotic spindle and its lagging during anaphase, which eventually leads to its elimination from the nucleus. However, the repetitive nature and rapid evolution of centromeres make their identification and comparative analysis across species and chromosomes challenging. Here, we used a combination of cytogenetic and genomic approaches to identify the centromeric sequences of two closely related songbird species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (L. luscinia). We found a 436-bp satellite repeat present in the centromeric regions of all regular chromosomes (i.e., autosomes and sex chromosomes), making it a strong candidate for the centromeric repeat. This centromeric repeat was highly similar between the two nightingale species. Interestingly, hybridization of the probe to this satellite repeat on meiotic spreads suggested that this repeat is missing on the GRC. Our results indicate that the change of the centromeric sequence may underlie the unusual inheritance and programmed DNA elimination of the GRC in songbirds.

Animals

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin C in BrdU-labeled human chromosomes.

The BrdU-Hoechst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs) induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combination of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significantly increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of SCE might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.

Bromodeoxyuridine