A deletion of the albsino ( c ) locus on mouse chromosome 7 has been demonstrated using Qand G-banding methods in a mouse heterozygous for the radiation-induced lethal albino allele, c Z ~ H . The deletion, which is thought to be 1-6 cM long, represents about 7.6% of the length of the metaphase chromosome. OMPLEMENTATION studies of a series of radiation-induced mutations at the albino ( c ) locus with multiple biochemical and ultrastructural effects led to the distinction of four different complementation groups among six lethal alleles, supporting the interpretation that these represent overlapping deletions of various sizes (GLUECKSOHN-WAELSCH et al. 1974). Homozygotes for each of the six alleles were lethal, four of them dying perinatally and two during early embryogenesis. No complementation was observed in combinations of any of the other five alleles with c2jH, which is thought to be the biggest deletion. The complementation studies included determinations of activity of the enzymes glucose-6-phosphatase, tyrosine aminotransferase and serine dehydratase, serum protein measurements, and examination of subcellular ultrastructure, viability, growth, fertility and morphogenesis (GLUECKSOHN-WAELSCH et al. 1974). The cZjH mutation was shown to be deleted at the structural locus fo r the enzyme mitochondrial malic dehydrogenase mod-2 ( ERICKSON, EICHER and GLUECKSOHN-WAELSCH 1974). Previous genetic studies attempting to determine the limits of possible chromosomal deletions in the radiation-induced c-alleles made use of two outside markers: taupe (proximally, 2% recombination with albino), and shaker-1 (distally, 4% recombination with albino). Both of these loci were found to be intact in c14c0s, c ~ ~ * " , PK, and c~~ (GLUECKSOHN-WAELSCH and CORI 1970). This work was supported by grants from the National Foundation-March of Dimes and the Naponal Institutes of Health (CA 12504, GM 18153 and HD 010193) and the American Cancer Society (VC M). O.J.M. is a Career Scientist of the Health Research Council of the City of New York. Genetics 78: 905-910 November. 1974. 906 D. A. MILLER et al. Although c"jH was not available at the time of these studies, it is assumed that this deletion, too, does not extend as far as the taupe locus in one direction or the shaker-1 locus in the other. The introduction of chromosome banding techniques greatly increased the accuracy of detection of cytological abnormalities in various mammalian species. However, few attempts have yet been made to apply banding techniques to the recognition of presumptive deletions, and the first of these was unsuccessful (DEV et al. 1971). Linkage group 1, which carries the c locus, was assigned to chromosome 7 in the mouse (KOURI et al. 1971; MILLER et al. 1971b; Committee 1972). We report here the presence of a visible deletion in chromosome 7 in mice heterozygous for PH. MATERIALS A N D METHODS A male mouse heterozygous for c*jH ( c 2 S I I / c ~ h ) was mated to two AKR females. The pregnant females, which were sacrificed on day 14 o r 15, produced nine albino (cZ55H/c) and three non-albino (cch/c) fetuses. The albino embryos were minced and pooled, and primary cultures were set up as described previously (MILLER et al. 1971a). Control cultures from non-albino littermates were set up in the same way. After two days in culture, mitotic cells were harvested by mild trypsinization to remove the loosely attached cells. The freed cells were transferred to a centrifuge tube containing a drop of Colcemid (10pg/ml) and spun at 600 rpm. The medium was replaced with 0.07 M KC1, left at room temperature for 15 minutes and then replaced, after centrifuging, with methanol : glacial acetic acid (3: 1). After three changes of fixative, the suspended cells were dropped onto chilled, wet slides, air dried, and stored at 4". Within 6 days some of the slides were stained with quinacrine mustard (.05 pg/ml) for eight minutes, rinsed with water and tris-maleate buffer at pH 5.6, and mounted in the same buffer. The slides were examined either by transmitted or incident UV light (HBO 200 watt mercury bulb illumination), BG 12 exciter filter and K530 barrier filter, photographed on H&W control film with 30-40 second exposures, developed in H&W developer and printed on Ilford paper. Other slides were stained for G-banding by the method of SUN, CHU and CHANG (1973) and photographic prints made. About 100 Qand G-banded metaphase prints were made on the albino embryos, and 42 karyotypes were prepared. About 40 prints were made of the normal embryos, and 12 karyotypes were prepared. The chromosomes 7 were measured on prints of 28 heterozygous and 10 normal metaphase cells using a ruler marked at 1/64-inch intervals. Each chromatid was measured twice and the four values were summed.

American Journal of Medical GeneticsVolume 57, Issue 3 p. 498-500 Letter to the Editor Pallister-Killian syndrome detected by fluorescence in situ hybridization Merlin G. Butler M.D., Ph.D., Corresponding Author Merlin G. Butler M.D., Ph.D. Departments of Pediatrics, Pathology, and Orthopaedics, Vanderbilt University School of Medicine, TennesseeDD-2205 Medical Center North, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232Search for more papers by this authorV. G. Dev, V. G. Dev Genetics Associates, Nashville, TennesseeSearch for more papers by this author Merlin G. Butler M.D., Ph.D., Corresponding Author Merlin G. Butler M.D., Ph.D. Departments of Pediatrics, Pathology, and Orthopaedics, Vanderbilt University School of Medicine, TennesseeDD-2205 Medical Center North, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232Search for more papers by this authorV. G. Dev, V. G. Dev Genetics Associates, Nashville, TennesseeSearch for more papers by this author First published: 3 July 1995 https://doi.org/10.1002/ajmg.1320570330Citations: 11AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume57, Issue33 July 1995Pages 498-500 RelatedInformation

We report the clinical evaluation of an improved DNA probe assay for the characteristic genetic marker of human CML, observed by cytogenetics and designated the Philadelphia chromosome (Ph1). The Ph1 chromosome results from the fusion of c-abl proto-oncogene sequences from chromosome 9 to phl gene sequence on chromosome 22. (The phl gene is often referred to as bcr. However, for clarity we prefer to reserve the designation "bcr" for the region within the phl gene in which translocation breakpoints have been found to occur. We also find it useful to distinguish between two such regions in phl, bcr-210 and bcr-190, named after the 210- and 190-kDa phl/abl fusion proteins resulting from translocations with breakpoints in the respective regions. We refer to the corresponding chromosomal translocations as Ph1(bcr-210) and Ph1(bcr-190).) DNA, extracted from peripheral blood (PB) or bone marrow (BM) and digested with restriction endonuclease BglII, is hybridized with a probe (phl/bcr-3) spanning a breakpoint cluster region within phl. Rearrangements are revealed by the presence of one or two novel junction fragments. Clinical specimens from leukemic patients with active disease were compared by cytogenetic and DNA probe analysis at seven centers in the United States and Europe. The probe assay identified the phl rearrangement in 190 of 191 cases of Ph1-positive CML, as well as in 12 of 27 clinically diagnosed CML specimens lacking a typical Ph1 chromosome. DNA rearrangements also were seen in two of six cases of Ph1-positive ALL. No false positive results were obtained among 93 non-leukemic controls. Mixing experiments showed that the DNA probe assay can detect as few as 1% leukemic cells in a specimen. A preliminary study of CML patients in remission after allogeneic BM transplantation revealed a small fraction of residual Ph1-positive leukemic cells in a significant number of such patients.

The use of two polyclonal activators, dextran sulfate (DxS) and lipopolysaccharide (LPS), with or without the presence of additional antigen, is presented here as a system for exploring the antibody response of normal (naive) amd primed B cells. This system expands populations of cells not normally observed under in vivo regulation. By fusing such unnaturally activated B cells, anti-p-azophenylarsonate hybrids were produced that secrete different isotypes of antibodies. The frequencies of isotopes expressed by these hybrids may correspond to the chromosomal order of the heavy chain genes because greater numbers of IgM- and IgG3-secreting hybrids were produced than IgG2a hybrids. Only one IgA hybrid was observed. When DxS and LPS were used to stimulate antigen-primed B cells, hybrids were generated that simultaneously secrete two isotopes of antibody. These hybrids may represent a model of the antigen-stimulated maturational class-switch step observed in normal B cells that involves the expression of IgM and IgG isotypes by the same cell. Such hybrids offer an opportunity to study antibody regulation and diversity by examining the rearrangement of genes during the Ig switch, by exploring the nature of the necessary transitions of mRNA transcription and translation to produce functional antibodies, and by probing the structure and specificity of such antibodies.

Establishment and characterization of an endometrial adenocarcinoma cell line are described. The tumor cells, designated AC-258, originated from a patient with a poorly differentiated adenocarcinoma of the endometrium. These cells have been passed in culture more than 102 times and have maintained their morphological and chromosomal integrity in a fashion suggestive of monocloning. Cells from passages 1, 58, and 102 were aneuploid. The average number of chromosomes per cell was 64.8, and 8–17 marker chromosomes per cell were identified and described. Histological evaluation of tumor explants from 3 passages grown in nude (athymic) mice revealed morphologic identity. The doubling time of AC-258 was 22 hr. No detectable estrogen- or progesterone-binding proteins were found. The AC-258 cell line provides researchers with another tool with which to investigate biochemical and biological properties of human cancer cells.

Active nucleolus-organizer regions (NOR) were identified on chromosomes 3, 4, 5, 7, 8, and 9 in a diploid Chinese hamster primary strain (CHFP-WG). However, they appeared only on one chromosome 4, both chromosomes 5, and the isochromosome for the short arm of chromosome 8 (iso 8p) in an established Chinese hamster line displaying gross chromosomal rearrangements (AK412). Some recognizable NOR sites, exhibiting active NOR in CHFP-WG cells fail to exhibit active NOR in AK412, suggesting that a specific suppression of NOR has occurred in the AK412 cells.

Cytogenetic analysis of a balanced reciprocal translocation t(5;13) carrier and her unbalanced 5p-conceptus was carried out. This carrier mother had previously given birth to a child with cri-du-chat syndrome. Silver staining demonstrated the breakpoint on 13p within the nucleolus organizer region (NOR). In the carrier, the NORs both at the original site 13p0 and at the translocated site (5p) were silver-stained, indicating that the rRNA genes at both sites were transcribed. The NOR at the derivative chromosome 5 was also silver-stained in the fetus.

Nucleolus organizer regions (NOR) are actively expressed on both sets of parental chromosomes in mouse-Chines hamster hybrid cells retaining 85–95% of each parental complement. A hamster NOR, which is active in primary fetal hamster cells, but suppressed in the established AK412 line, is reactivated in these hybrids, demonstrating control of NOR expression.

Banded karyotypes were prepared for three independent sublines of clone 745A, a Friend virus-induced erythroleukemia cell line originally produced by Dr. C. Friend. Each subline had the same chromosome complement, with a near-diploid number of chromosomes, two-thirds of which appeared normal. The remaining one-third were structurally rearranged marker chromosomes, including eight whose origins could be determined from their banding patterns. A fourth subclone of line 745A, under different selection pressure, showed changes in five marker chromosomes but no changes in the normal chromosomes, suggesting that the markers contained unstable sites, perhaps viral integration sites. Another Friend virus-induced erythroleukemia cell line, FSD-1/F4, developed independently by Dr. W. Ostertag, had a near-diploid number of chromosomes, about one-fourth of which were abnormal chromosomes. The only similarity between these markers and those in subclones of 745A, other than participation of some of the same chromosomes in centric fusion, was the inclusion of the proximal two-thirds of chromosome 15 in one of the markers of each line. This adds to the number of mouse tumor cells or cell lines in which No. 15 is altered or duplicated. In FSD-1/F4 cells there was a striking increase in the size of the secondary constriction of one of the two chromosomes 19, suggesting that the rRNA genes on this chromosome had been amplified.

Chromosomes with active nucleolus organizer regions (NORs) were identified by combined Q-banding, C-banding (in some cases), and silver staining in mouse cell lines. NOR-bearing chromosomes were overrepresented among the chromosomes involved in Robertsonian translocations in LM(TK-), A9, and RAG cell lines. Usually only one NOR-bearing chromosome was seen in any biarmed chromosome; relatively few contained two NOR-bearing chromosomes. Thus the nucleolus plays an important role, but nucleolar fusion is relatively unimportant, in the origin of Robertsonian translocations in the mouse.

The direction of chromosome loss in two sets of mouse-Chinese hamster hybrids was compared with the direction of segregation of the same hybrids, to which an additional X chromosome derived from either of the mouse sarcoma lines MethAa, MethAs, or CMS4, was introduced at the time of the fusion. The addition of the X chromosome was carried out by substituting in place of the Chinese hamster parent a mouse X containing microcell hybrid of the latter. It was found that the addition of an X chromosome reverses the direction of chromosome segregation, but it can do so only if the mouse parent in the hybridization is different from the line from which the X originated. The possible reasons for recognition by the cells of a native and a foreign X are discussed. The existence of a multigene family on the X chromosome, involved in this recognition, is proposed.

A human fibrosarcoma line, HT1080-6TG, with a near diploid number of chromosomes, has an average of 7.3 chromosomes with an Ag-stained nucleolus organizer region (NOR). Cells of this line with an increased number of chromosomes have an increased number of Ag-stained NORs. This cell line has been used as the human parent in constructing mouse-human and rat-human hybrids that segregate rodent chromosomes. The hybrid ccell lines, which have 100 or more chromosomes per cell, show a proportionate increase in the number of Ag-stained NORs (means, 11.4--16.8). The frequency of association of acrocentric chromosomes increases in a similar fashion. There is no evidence of inactivation of human NORs in these cells.

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.

The binding of highly purified anti-nucleoside antibodies to mouse (Mus musculus) metaphase chromosomes was studied by an immunofluorescence technique. The chromosomal DNA was denatured by one of two selective denaturation procedures because these antibodies reacted with single stranded but not native DNA. After ultraviolet irradiation (UV), which produced single stranded regions primarily in AT rich DNA, the binding of antiadenosine (anti-A) produced a pattern of fluorescent bands similar to that produced by quinacrine (Q-bands). Additional foci of bright fluorescence were observed at the centrometric (C-band) regions, which are known to contain AT rich satellite DNA. After photooxidation, which produced single stranded regions in GC rich DNA, the binding of anti-A produced a fluorescent banding pattern similar to the R-banding pattern seen after thermal denaturation and staining with coriphosphine O. After photooxidation, R-band patterns were also obtained with anti-cytidine (anti-C) and anti-5-methylcytidine (anti-M). After either UV irradiation or photooxidation, anti-M, but not anti-C, showed intense binding to the C-band regions of mouse chromosomes. — These findings led to the following conclusions: (1) Antibody banding patterns reflect the presence of a class of AT rich, GC poor DNA in chromosome regions which show bright quinacrine fluorescence and in the regions that contain the AT rich satellite DNA. (2) The alternate, quinacrine dull regions contain a relatively GC rich class of DNA which appears to be more highly methylated than the AT rich DNA in the Q-bright bands, but not the AT rich satellite DNA in the Q-dull C-bands. (3) 5-Methylcytosine residues occur in a sequence of mouse satellite DNA that contains both adjacent pyrimidines and guanine residues. The basic repeating unit of mouse satellite DNA is known to contain the sequence 5′-GAAAAATGA-3′ (Biro et al., 1975). Therefore, assuming the antibodies used could detect single bases in denatured DNA, the methylated sequence in mouse satellite DNA \({\text{could be }}\begin{array}{*{20}c} {5\prime - {\text{A M G AAAAA T GA - 3}}\prime } \\ {3\prime - {\text{T G M TTTTT A C T - 5}}\prime } \\ \end{array} {\text{ or }}\begin{array}{*{20}c} {5\prime - {\text{G AAAAA T G A M G AA - 3}}\prime } \\ {3\prime - {\text{C T T T T T AC T G M TT - 5}}\prime } \\ \end{array} .\)