Localization of the human haptoglobin genes distal to the fragile site at 16q22 using in situ hybridization

The distamycin A-sensitive fragile site fra(16)(q22) is a precisely localized chromosomal marker. When expressed at metaphase, it visibly separates the chromosome material on either side of the fragile site. Using a cDNA probe encoding both the alpha and beta haptoglobin chains, the haptoglobin loci were found by in situ hybridization to be distal to fra(16)(q22).     

Induction of distamycin A-inducible rare fragile sites and increased sister chromatid exchanges at the fragile site

Summary Expression of distamycin A-inducible rare fragile sites by AT-specific DNA-ligands was examined in lymphoblastoid cell lines derived from heterozygous carriers for the fra(8)(q24), fra(16)(pl2), and fra(16)(q22) sites. The sensitivity of fragile site expression to the inducers was different at these fragile sites. The expression of fra(8)(q24) was induced markedly by Hoechst 33258, but not by distamycin A or berenil. An increased expression of fra(16)(p12) was found following treatment with Hoechst 33258 or berenil, but not with distamycin A. At fra(16)(q22), distamycin A markedly induced the fragile site, but Hoechst 33258 and berenil did not. Since their response to the different inducers was similar to that found in cultured lymphocytes, lymphoblastoid cell lines appear to retain their inherent properties. Although BrdUrd alone did nto induce any fragile sites, concomitant treatment with BrdUrd plus the inducer was synergistically effective in inducing all the fragile sites. An increased frequency of sister chromatid exchanges was observed at fra(16)(p12) following simultaneous treatment with BrdUrd and berenil, mainly when the site was expressed as an isochromatid gap. Thus, the induced fra (16)(pl2) site is a hot spot for the formation of sister chromatid exchanges, as found in other reported fragile sites.     

Familial fragile site found at the cancer breakpoint (1)(q32)

Summary A normal baby was cytogenetically examined immediately after birth for the possible presence of a fragile (16)(q22), which had been found in her mother and in her retarded sister with a 46,XX;46,XX,del(16)(q22) mosaic karyotype. Distamycin a was added to the cultures to enhance the fragile (16)(q22) expression. The response of the baby to the action of distamycin a in vitro was much greater than that of her family members. A fragile (16)(q22) was induced in many cells as well as a fragile (1)(q32), which was also found in her mother. This fragile site, which is known to be a cancer breakpoint, has not been reported so far either to be familial or to be inducible by distamycin A. The concomitance of fragile (1)(q32) with fragile (16)(q22) and their possible significance are considered.     

Heritable fragile sites on human chromosomes. XI. Factors affecting expression of fragile sites at 10q25, 16q22, and 17p12.

The fragile sites at 10q25, 16q22, and 17p12 can all be induced in lymphocyte culture by BrdU or BrdC added 6-12 hrs prior to harvest. Without induction, fra(10)(q25) is rarely expressed spontaneously, whereas fra(16)(q22) is frequently expressed spontaneously. Fra(17)(p12) is frequently expressed spontaneously but is probably expressed only after induction in some individuals. Distamycin A, netropsin, and Hoechst 33258 induced high levels of expression of fra(16)(q22) and fra(17)(p12) but did not enhance expression of fra(10)(q25). The mechanisms of induction of fra(16)(q22) by BrdU and distamycin A appear to be different, since the time of induction by BrdU reaches a maximum about 12 hrs prior to harvest whereas induction by distamycin A requires much longer exposure. The fragile sites at 10q25 and 16q22 were both induced in fibroblast culture by BrdU. Fra(17)(p12) is accepted as a fragile site because preliminary studies show that it behaves similarly in lymphocyte culture to fra(16)(q22); however, there is only limited evidence for fragility at 17p12.     

Population cytogenetics of rare fragile sites in Japan

Summary A population cytogenetic study of three groups of rare fragile sites defined in Human Gene Mapping 8 (HGM8, Berger et al. 1985) has been conducted using peripheral blood lymphocytes of healthy Japanese subjects. We have examined 1,022 blood donors for folate-sensitive and bromodeoxyuridine (BrdU)-requiring, and 845 for distamycin A-inducible fragile sites. Out of 17 rare autosomal fragile sites defined in HGM8, the following six were identified in Japan; folate-sensitive fra(2)(q11), fra(11)(q13) and fra(11)(q23), distamycin A-inducible fra(16)(q22) and fra(17)(p12), and BrdU-requiring fra(10)(q25). The incidences of distamycin A-inducible fra(16)(q22) (1.42%) and fra(17)(p12) (3.08%) were considerably higher than those of the other sites in Japan. Furthermore, a folate-sensitive fra(17)(p12) and a distamycin A-inducible fra(8)(q24.1) have been newly found in the present study. Their incidences were 0.10% (1/1,022) and 0.71% (6/845), respectively. Since the expression of this fra(17)(p12) was induced by fluorodeoxyuridine, supressed by thymidine, but not induced by distamycin A, it can be classified as a folate-sensitive site. The expression of the new distamycin A-inducible fra(8)(q24.1) was also enhanced by treatment with Hoechst 33258, berenil and 4,6-diamidino-2-phenylindole (DAPI). This fragile site fulfils all four classical criteria suggested by Sutherland (1979) and also new criteria for a rare fragile site defined in HGM8 (Berger et al. 1985).     

Fragile chromosome 16(q22) cause a balanced translocation at the same point

A father with a fragile 16(q22) has a son with a de novo balanced translocation 1;16. Both the fragile site and the break point at chromosome 16 are similar (q22). The question of whether the fragile site can cause a structural chromosome abnormality at the same point is discussed.     

A mouse-human hybrid cell panel for mapping human chromosome 16

A mouse-human hybrid cell panel for human chromosome 16 was constructed from human cell lines with breakpoints on chromosome 16 at p13.11, q13, q22 and q24. Fusions with the human fibroblast line GM3884, t(X;16)(q26;q24) allowed the isolation of clones with either the derivative X or the derivative 16 as the only human chromosome. This was a consequence of both the genes APRT and HPRT being involved in the translocation. The breakpoints of the line GM3884 were confirmed by aphidicolin induction of the common fragile site at 16q23. The results of the fusions with this line suggest a localisation of the APRT gene at 16q24 and confirm the localisation of HPRT to Xq26 to Xq27.3. These hybrid cell lines enable the localisation of genes and DNA fragments to six clearly defined regions. Further localisation within three of these regions is possible by use of the three fragile sites on chromosome 16. In situ hybridisation with the probe pBLUR confirmed that of three lines tested all contained a single human chromosome.     

A comparison of constitutive heterochromatin staining methods in two cases of familial heterochromatin deficiencies

Summary Using DAPI staining after pretreatment with distamycin A we detected a familial deficiency of chromosome 16 heterochromatin. A distinct positively staining band, however, was seen after C-banding. Thus, by using these different heterochromatin staining methods, heterogeneity of the constitutive heterochromatin in the centromeric region of human chromosome 16 was indicated. The same C-banding procedure was also applied to a previously described familial deficiency of chromosome 9 heterochromatin evidenced using distamycin A/DAPI staining and G 11 staining (Buys et al., 1979). In this case a C-band appeared to be virtually absent on the relevant chromosome. These staining methods may be valuable tools in the study of chromosome polymorphisms.     

Localization of the human GM-CSF receptor beta chain gene (CSF2RB) to chromosome 22q12.2-->q13.1.

The gene for the beta-chain of the human GM-CSF receptor (CSF2RB) has been mapped to chromosome 22 by PCR analysis of a series of human x rodent somatic cell hybrids. In situ hybridization to normal human chromosomes and two translocations involving chromosome 22 and the chromosome expressing the rare fragile site FRA22A place the gene in the region 22q12.2-->q13.1, proximal to the fragile site.     

Autosomal fragile sites

It is possible to distribute the 17 autosomic fragile sites presently known in three categories according to their sensitivity: BrdU-sensitive sites (10q25, 16q22, 17p12), distamycin A-sensitive sites (16q22, 17p12) and folate- and thymidilate-sensitive sites (2q11-q14, 3p14, 6p23, 7p11, 8q22, 9p21, 9q32, 10q23, 11q13, 11q23, 12q13, 16p12, 16q23, 17p12, 20p11). Four fundamental problems are discussed, first the relation between the presence of a fragile site and the phenotype, secondly the incidence of autosomic sites, third the origin of fragility (particularity of DNA structure, defect of the DNA/proteins binding and abnormal arrangement of chromatin, abnormality of the metaphasic scaffold) and fourth the localization of fragile sites.     

Role of heterochromatin during preferential 9q;22q translocation in chronic myelogenous leukemia

The secondary constriction region (h) of human chromosome 9 was evaluated in 55 chronic myelogenous leukemia (CML) patients with respect to its size and position. Each case was examined by C-banding and distamycin A-4,6-diamidino-2-phenylindole techniques for the expression of the h regions. When one h region of chromosome 9 was larger, it was more frequently involved in the reciprocal translocation with chromosome 22. In addition, there was a higher incidence of pericentric inversions in the h regions in the translocated chromosome 9 when compared with normal homologues. The role of the constitutive heterochromatin of chromosome 9 as a possible influencing factor during 9q;22q translocation in CML is suggested.     

The fragile site (16)(q22)

Summary In the lymphocytes of heterozygous carriers of the rare autosomal fragile site (16)(q22) an exceptionally high frequency of sister chromatid exchanges was demonstrated at the induced fragile site by means of simultaneous berenil and BrdU treatment of the cultures. The rate of sister chromatid exchanges at q22 is also increased in the fragile chromosome 16 by treating the cells with BrdU alone. The possible reasons for the preferential occurrence of induced and spontaneous sister chromatid exchanges at fra (16)(q22) are discussed.     

Segregation analysis of rare autosomal fragile sites

Summary Segregation analyses were performed on pedigrees with rare autosomal fragile sites. The results of the analysis of pedigrees with folate sensitive fragile sites, including 2q1, 6p23, 7p11, 8q22, 9q32, 10q23, 11q13, 11q23, 12q13, 16p12, and 20p11, suggested that expression of the gene depended on the carrier parent: it was only 50% penetrant when transmitted by a carrier father, but fully penetrant when transmitted by a carrier mother. Pedigrees with the bromodeoxyuridine (BrdU) fragile site, fra(10)(q25), showed the same trend but the results were not statistically significant. In addition, 38 of the 44 probands with folate sensitive or BrdU-sensitive fragile sites received the gene from their carrier mother and only six received it from their father. In contrast, the analysis of pedigrees with the distamycin A-inducible site, fra(16)(q22), gave the results expected for a simple codominant trait with complete penetrance. Probands with this fragile site received the gene equally from mothers or fathers. The genetic implications of these results are discussed.     

Demonstration of Y/autosomal translocations using distamycin A

Summary The condensation of the brightly fluorescing Y-heterochromatin is prevented by cultivating leukocytes in a medium containing distamycin A. This technique provides a reliable method for the identification of those Y/autosomal translocations in which Y heterochromatin is involved. A case of a familiar Y/22 translocation and the distamycin A technique are described.     

Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A

A DNA-binding AT-specific oligopeptide antibiotic, distamycin A, was used as non-fluorescent counterstain in conjunction with the DNA-binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) to investigate the effect of the antibiotic on DAPI fluorescent banding of human chromosomes. Distamycin A-pretreated metaphases and interphase nuclei exhibited a significantly lower overall fluorescence intensity than DAPI controls. Chromosome arms were pale and intercalary DAPI bands (Q bands) were obliterated, but some specific regions of constitutive heterochromatin remained brightly fluorescent. These were mainly the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y. The distamycin A/DAPI banding pattern appears to be comparable to that reported for anti-5-methylcytosine binding [11]. The observations are discussed as they relate to the roles of chromosomal DNAs and proteins in chromosome banding.     

The fragile site (16) (q22)

Summary The rare fragile site at 16q22 was experimentally induced in lymphocyte cultures with various AT-specific, non-intercalating DNA-ligands. The optimum conditions for the induction of fra (16)(q22) were determined. The best expression of fra (16)(q22) was found with the aromatic diamidine berenil which is recommended for further studies on this fragile site. The results indicate that fra (16)(q22) is a region with AT-rich, late replicating DNA. The simultaneous treatment of lymphocytes with berenil and aphidicolin (inhibitor of DNA polymerase ) induces both the rare fra (16) (q22) and the common fra (16) (q23) within the same chromosome. A population study on 350 unselected individuals showed that fra (16)(q22) is the most common of all rare autosomal fragile sites in man. The frequency of individuals heterozygous for fra (16)(q22) is 5.1% no homozygosity for fra (16) (q22) was detected. Statistical analysis indicates that the population is in Hardy-Weinberg equilibrium with respect to the fragile and non-fragile chromosomes 16.     

The fragile site on chromosome 16 (q21q22)

Summary The significance of the fragile site on 16 (q21q22) has not yet been fully evaluated. New data will contribute to the understanding of this cytogenetic finding. Therefore we report on four families where a chromosome 16 with fragile site was segregating and such problems as infertility, abortions, malformations, and ancuploidy were present. The hypothesis that this fragile site is a site of viral modification (or integration?) is considered.     

A refined physical map of the long arm of human chromosome 16

Mapping of 33 anonymous DNA probes and 12 genes to the long arm of chromosome 16 was achieved by the use of 14 mouse/human hybrid cell lines and the fragile site FRA16B. Two of the hybrid cell lines contained overlapping interstitial deletions in bands q21 and q22.1. The localization of the 12 genes has been refined. The breakpoints present in the hybrids, in conjunction with the fragile site, can potentially divide the long arm of chromosome 16 into 16 regions. However, this was reduced to 14 regions because in two instances there were no probes or genes that mapped between pairs of breakpoints.     

Berenil-induced undercondensation in human heterochromatin

The aromatic diamidine berenil specifically inhibits the condensation of a subset of constitutive heterochromatin in human lymphocyte cultures. In the normal male chromosome complement, only the quinacrine-brilliant Y heterochromatin exhibits distinct undercondensation. The optimal culture conditions for inhibiting heterochromatin condensation are achieved when berenil is added at a final concentration of 150 micrograms/ml 24 h before cell harvest. Various examples of the use of berenil in the analysis of chromosome rearrangements involving quinacrine-brilliant heterochromatin are presented. A variant, giant-satellited chromosome 22 was found to respond to berenil treatment, although its enlarged and quinacrine-bright short-arm region did not contain Y heterochromatin. Southern blot analysis and chromosome in situ hybridization suggested that most chromosome 22 variants do not stem from Y; acrocentric translocations. The experimentally undercondensed Y heterochromatin is characterized by moderate C-band labeling, bright quinacrine fluorescence, and specific silver staining. At the ultrastructural level, undercondensation is associated with loosely packed, mutliply folded chromatin fibers with a diameter of approximately 250 A and organized probably as loops.     

Human zinc finger gene ZNF23 (Kox16) maps to a zinc finger gene cluster on chromosome 16q22, and ZNF32 (Kox30) to chromosome region 10q23-–q24

Two members of the KOX gene family, ZNF23 (KOX16) and ZNF32 (KOX30), have been mapped by in situ hybridization to chromosome regions 16q22 and 10q23-q24, respectively. The map location of ZNF23 and ZNF32 placed these zinc finger protein genes near to chromosome loci that, under certain in vitro conditions, are expressed as fragile sites (FRA16B, FRA16C) and (FRA10D, FRA10A, FRA10B and FRA10E). Human zinc finger gene ZNF32 maps to a chromosome region on 10q23-24 in which deletions have been observed associated with malignant lymphoma on 10q22-23 and with carcinoma of the prostate on 10q24. ZNF23 is located on 16q22 in a chromosomal region that has been involved in chromosome alterations characteristic of acute myeloid leukemia. A second Kox zinc finger gene (ZNF19/KOX12) was recently mapped to the same chromosome region on human chromosome 16q22. In the analogous murine position, the murine zinc finger genes Zfp-1 and Zfp-4 are found in the syntenic 16q region of mouse chromosome 8. Thus, ZNF19 and ZNF23 might be members of an evolutionarily conserved zinc finger gene cluster located on human chromosome 16q22.