Localization of cloned human DNA sequences and analysis of chromosomal alterations bv in situ hvbridization Patracchini P, Aiello V, Marchetti G, Palazzi P, Ferrati M, Calzolari E, Bernardi F. Localization of cloned human DNA sequences and analysis of chromosomal alterations by in situ hybridization. Liver 1992 (Spec. issue) 12: 280-285.

Abstract: The in situ hybridization technique was used for the localization on human chromosomes of single-copy and repeated sequences and, in addition, for the characterization of altered human chromosomes. Two anonymous clones, single or low-copy, obtained from a human X chromosome library were localized on the distal part of the long arm and in the paracentromeric region of X chromosome, respectively. A genomic fragment of the single-copy thyroglobulin (TG) gene was used to confirm the localization on the distal part of the long arm of chromosome 8. The localization and distribution on human chromosomes of the glyceraldehyde-3-phosphate dehydrogenase (GAPD) multigene family obtained by in situ hybridization and by somatic cell hybrids were compared. A phosphoglycerate kinase (PGK) c-DNA clone, which detects genic and pseudogenic sequences on the X chromosome, was used for the characterization of three small ring markers present in unrelated female patients.

Introduction

In situ hybridization of metaphase and prometaphase chromosomes has been used for the localization of single, low and multi-copy DNA sequences in eukaryotes and particularly in humans (1). In situ hybridization has extended and improved the information obtained with other physical mapping methods such as somatic cell hybrid panels and pulsed-field gel electrophoresis. The chromosomal localization of cloned anonymous DNA fragments and of single, low and multicopy genes have been used to define genetic maps (2), useful genetic markers (3), and for comparative studies on structure and evolution of chromosomes (4). The molecular diagnosis of chromosomal abnormalities and the localization of breakpoints have also been obtained (5-7), thus identifying regions involved in chromosomal rearrangements and complementing conventional cytogenetic analysis. By means of in situ hybridization we have previously sublocalized single-copy sequences: protein C gene on 2q13-ql4 (8), von Willebrand factor pseudogene on 22qll.22-qll.23 (9) and two human phosphatidylinositol 3-kinase homologous se280

P. Patracchini, V. Aiello*, 6. Marchetti, P. Palazzi", M. Ferrati, E. Calzolari" and F. Bernardi Centro di Studi Biochimici delle Patologie del Genoma Umano, lstituto di Chimica Biologica and 'Istituto di Genetica Medica, Universita di Ferrara, Ferrara, Italy

Key words: In sit0 hybridization; thyroglobulin gene; anonymous DNA sequences: glyceraldehyde-3-phosphate dehydrogenase; phosphoglycerate kinase; multigene family; chromosomal mapping; ring marker chromosomes Bernardi Francesco, Centro di Studi Biochimici delle Patologie del Genoma Umano, lstituto di Chimica Biologica Universita di Ferrara, Via L. Borsari, 46. 1-44100 Ferrara, Italy. Phone: 39/532/210232. Fax: 39/532/202723 Accepted for publication 21 February, 1992

quences on 5q12-ql3 and 19q13.2-q13.4 (10). We have also used this technique to study a complex autosomal translocation involving chromosomes 8, 13 and 15 (11). In order to exemplify possible approachs with the in situ hybridization technique we report experiments for the localization of anonymous human DNA sequences and of specific, single and reiterated, human genes. Moreover, studies to established the origin of ring marker chromosomes are reported. Material and methods

In situ hybridization experiments were performed in accordance with Harper and Saunders (12) with some minor modifications. A detailed description of the technique has been recently reported by Naylor et al. (13). Metaphase spreads

Metaphase spreads were from peripheral blood of normal male and female subjects and of 3 unrelated female patients (cases 108, 117 and 162) with the following karyotypes: case 108, 45,X/46,X +

Chromosomal mapping by in situ hybridization marker (mosaic 50%); case 117, 45,X/46,X+ marker (mosaic 50%); case 162, 45,X/46,X+ marker (mosaic 16"Ao). At the cytogenetic analysis the observed markers were suggested to be X derivative. Mitoses were prepared with standard procedures, also used for routine analysis of human chromosomes (13). After spread, the metaphases were checked with phase-contrast optics and the selected slides were stored in a dessicator.

The slides were dipped in Ilford L4 nuclear track emulsion diluted 1:1 with 1% glycerine and exposed for 4 wk at 4°C in a light-proof box. Exposed slides were developed for 4 min, fixed for 5 min, extensively rinsed in tap water and air dried.

Pretreatment of the spreads

G-chromosome banding and analysis of grain distribution

The slides were treated with RNaseA at a concentration of 100 pg/ml in 2 x SSC (0.3 M NaCI, 30 mM sodium citrate) at 37°C for 1 h. The slides were rinsed in 2 x SSC for 30 rnin at room temperature. The chromosome preparations were then acetylated in 0.1 M triethanolamine pH 8.00/acetic anhydride 0.5%, dehydrated for 2 rnin each in 50, 70, 90 and 100% ethanol and air-dried.

The slides were immersed in a pH 6.8 phosphatebuffered Wright's stain (3:l) for 5-10 min, then briefly rinsed in running tap water and air-dried (14). Metaphase spreads with a limited extrachromosoma1 background and a good chromosome morphology were selected; only grains in direct contact with the chromosomes were scored; clusters of grains were considered as a single hybridization event. The silver grain distribution was determined by analysis of photographed spreads under direct microscope observation. Labelings were located and assigned to chromosome bands following the international nomenclature (ISCN 1978).

Chromosomal denaturation

Chromosomal DNA was denatured in 70% formamide/ 1 mM EDTA/2 x SSC pH 7.00 with HCl at 70°C for 4 min, followed by dehydration through ethanol series and air drying. Hybridization

The hybridization reaction mixture contained 3H dCTP labeled DNA probe (10' cpm/ng, 5-10 ng/ ml) in 50% formamide (v/v), 2xSSC, 1 x Denhardt solution (0.02% bovine serum albumin, 0.020/0 Ficoll and 0.020/0 polyvinylpyrrolidone), 10% dextran sulfate (w/v) and 250-300 pg/ml of yeast RNA. Seventy microliters of hybridization mixture were added to a selected area of the chromosome spreads and hybridization was performed at 37°C for 18-20 h in a closed Petri dish. Rinses

After hybridization, slides were washed three times for 5 rnin each in 50% formamide/2 x SSC at 39"C, followed by three washes, 5 rnin each, in 2 x SSC at 39°C and 10 washes at room temperature for 3 h. In the GAPD in situ hybridization experiment three washing temperatures were used: 39"C, 42°C and 45°C. The slides divided into three groups were washed three times (10 min each) in 50% formamide/2 x SSC and three times (10 min each) in 2 x SSC. The slides then underwent 15 washes at room temperature for 4 h.

After rinses chromosome preparations were dehydrated and air-dried. Autoradiography

Probes DXS167 and DXS168

A human X chromosome library (1 5 ) was screened for single-copy clones. Two clones, DXS167 and DXS168, were further characterized (16). DXS167 contained an EcoRI fragment of 7.8 Kb and DXS168 presented two EcoRI inserts of 2.7 and 3.1 Kb. Both genomic clones were used on metaphases from normal male and female subjects. Thyroglobulin

A 2.5 Kb EcoRI-Hind111 genomic fragment (17) was used as probe on metaphase spreads from a normal male subject. Glyceraldehyde-3-phosphate dehydrogenase

The 1.2 Kb pcD4 clone isolated from a human fibroblast cDNA library (18) was used as probe on metaphases from normal males and females. Phosphoglycerate kinase

Metaphase spreads from 3 female patients carrying small ring markers were hybridized to a full-length 281

Patracchini et al.

............ .... ......

. u

4

X

X

Fig.2. Localization of the DXS168 clone. Distribution oflabeling on X chromosome.

Fig. I. Localization of the DXS167 clone. Distribution of labeling on X chromosome.

PGK cDNA clone (19) encoding the complete protein plus 3’ and 5’ untranslated regions. Results and discussion Localization of the DXS167 and DXSl68 clones

Preliminary localization of DXS 167 and DXSl68 clones, obtained from a human X chromosome library (1 5), was performed by “dot spot” hybridization of DNAs from individuals carrying normal and altered X chromosomes (16). The analysis of hybridization intensity indicated that DXS 167 was localized on the X long arm and that DXS168 was present on both arms. A detailed localization was investigated by in situ hybridization on subjects with normal karyotype. Using DXS167 as probe we analyzed 55 mitosis and 70 silver grains were detected (1.3 grains per cell). Fig. 1 is a schematic diagram of the 19 labels (27%) observed on X chromosome; 10 (52.6%) were detected in Xq27-28, thus indicating that the anonymous sequence DXS167 is localized on the distal part of the long arm. In this chromosomal region several disorders have been assigned (hemophilia A and B, adrenoleukodystrophy, glucose-6phosphate dehydrogenase deficiency, Emery-Dreifuss muscular dystrophy, color blindness, incontinentia pigmenti 2, fragile X mental retardation). No restriction fragment length polymorphism has been identified by several restriction enzyme digestions. In in situ hybridization experiments with DXSl68 clone 58 metaphase spreads were analyzed and 46 labels (46/257, 18%) were on X chromosome (Fig. 2). Grains were observed in X l p (16/ 46) and Xlq (16/46). Significant labels were also detected on the telomers of chromosomes 6 and 1 1. Since DXS 168 clone recognized DNA sequences in Southern blots additional to those contained in the 282

clone, the presence of two homologous sequences on X chromosome and of additional sequences on autosomes can be suggested. Sublocalization of thyroglobulin gene

Thyroglobulin is a very large dimeric glycoprotein (MR 660 000) synthetized in the thyroid gland of all chordates, it is the site of formation of the thyroid hormones and the substrate for the transport of iodine. Studies on the gene organization (20) provide probes for further analysis of the chromosomal localization. We report a detailed description and additional analysis of the TG gene sublocalization previously obtained (21) using a 2.5 Kb genomic probe (17). Fig. 3A shows the distribution of the grains detected on chromosome 8. Ninety-three spreads have been analyzed; 28 (27%) out of 103 labels were located on chromosome 8: 20 (7 1.4%) on the terminal part of the long arm, 8 (28.5%) on the short arm. A labeled metaphase spread is reported in Fig. 3B. The data confirm that TG gene is located in 8q24 in accordance with other authors (22-24), who have used different probes. ‘A minor labeling is present in 8p23-p22, probably due to a partial homology to other DNA sequences, as suggested by Southern blot hybridization performed in less stringent conditions (data not shown). The 8q24 region contains the human oncogene c-myc and is involved in translocations associated with Burkitt lymphoma (25). The analysis of polymorphic markers associated to thyroid or extrathyroid pathologies could be improved by a detailed mapping of the region. Localization of GAPD-related gene family

The glycolytic enzyme GAPD (EC 1.2.1.12) is a tetramer of four subunits that is expressed in all

Chromosomal mapping by in situ hybridization 8

Table 1. Localization of glyceraldehyde-3-phosphate dehydrogenase related sequences by means of in situ hybridization and somatic cell hybrids “In situ” hybridization (pcD4 probe)

Somatic cell hybrids (spMG3 probe) (33)

1p36.3-p35 1p l 3 . 3 q l 2 1q41.1 q 4 4

1

2p25.3-p23 2pl1.2ql2 2q3-37.3 3q27q29 4q32q35 5q33.1q35.3

4

6 q l 1 . 2 44 6q25q27

6

7~221515.2 7q33q36

7

9q34.1q34.3 1l p l 2 q 1 2

11

‘1 2~13.3-pl2.1 12.1 1.2q13.2 12q23q24.33

12

13q32q34

13

18q22q23

18

20ql3.1q13.2 xp22.3-p22.1 *Xpl 1.4q12.2 Xq21.2q22.3 Xq26-q28

XP

The asterisks indicate the sites of the functional gene (12pl3.31-pl3.1) and of a pseudogene ( X p Z l - p l l ) previously detected.

Fig. 3. Localization of TG gene. A) Distribution of silver grains on a schematic diagram of chromosome 8. B) Autoradiograph of a hybridized metaphase spread.

cells. The human amino acid and nucleotide sequences have been determined. Only one form exists and two different mRNA sequences have been characterized in human liver (18). Data from Southern blot experiments of human and somatic cell hybrid DNAs (18, 26) suggested the presence of a high number of GAPD-related sequences in the human genome. The localization and distribution of these sequences on human chromosomes reported (27) were further investigated by in situ hybridization in three different exDerimental conditions (see methods). Due to the high number of GAPD homologous sequences in the human genome a statistical analysis (28) of the grains distribution was performed and 25 sites were assigned (listed in Table 1). The previous localization of a functional gene in

1 2 ~ 1 3(29, 30) and of a pseudogene in Xpll-p21 (31, 32) are confirmed in our experiments. Using as probe the 3‘ portion of the GAPD cDNA in Southern blots from human rodent somatic cell hybrids, Benham and Povey (33) have mapped GAPD-related sequences on several chromosomes (1, 2q, 4, 6, 7, 8, 11, 12, 13, 15, 18, Xp and Xq) (Table 1). The presence of GAPD-related sequences on these chromosomes was also confirmed in our experiments, except the localizations on chromosomes 15 and 8, on which significant hybridization was observed in two experimental conditions.

Table 2. In situ hybridization of chromosome markers with a phosphoglycerate kinase probe Localization of grains

Case 108 Case 117 Case 162

Metaphases

X chromosome

Xq13

markers

18 29 47

14 12 21

9 7 11

6 4 5

483

Patracchini et al. PGK (EC 2.7.2.3), a mayor enzyme in glycolisis, is encoded by a single locus (PGK 1) on Xq 13. Two pseudogenes on Xq12 (PGKlPl) and on 6p21-ql2 (PGKlP2) and a related DNA sequence (PGK2) on 19pter-pl3.1 have been detected (37-39). The hybridization data using a PGK probe obtained in the analysis of three ring markers are summarized in Table 2. A significant number of grains were observed as expected on Xq13 and on the ring chromosomes in all cases. Fig. 4 shows labels detected on normal X and ring chromosomes. The number of grains detected on ring chromosomes is consistent with the presence of mosaicism and confirms the X origin of the markers. Acknowledgments This work was supported by P. F. Ingegneria Genetica and Biotecnologie CNR; by Ric Fin Reg Emilia Romagna and Veneto and by MURST.

References Fig. 4. In situ hybridization of ring marker chromosomes with a PGK c-DNA. Labels on the X and the marker chromosomes are shown.

Sequences on chromosomes 14, 16, 17, 19, 21, 22 and Y were not detected in either experiment. The number of total sites detected is in agreement with previous estimates (34, 35). The precise localization of several GAPD-related sequences obtained by in situ hybridization provides useful information for genome mapping and for the detection of human chromosomes or their fragments present in somatic cell hybrids and in radiation hybrid cell lines. Characterization of ring marker chromosomes

Constitutional and somatic chromosomal abnormalities are often identified by standard cytogenetic studies. However, the morphological analysis of chromosomes sometimes does not allow their characterization. Rings are altered chromosomes arising from breaks occurring on the either side of the centromere and subsequent rejoining of the broken ends (36). The origin of small ring markers is often difficult to identify by standard cytogenetic analysis. In order to characterize and confirm the origin of ring markers observed in 3 unrelated girls referred for Turner’s phenotype we performed in situ hybridization experiments using a PGK probe (19). 284

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Localization of cloned human DNA sequences and analysis of chromosomal alterations by in situ hybridization.

The in situ hybridization technique was used for the localization on human chromosomes of single-copy and repeated sequences and, in addition, for the...
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