assignment of three gene loci (pgk, hgprt, to the long … · the intrachromosomal localization of...

ASSIGNMENT OF THREE GENE LOCI (PGK, HGPRT, G6PD) TO THE LONG ARM OF THE HUMAN X CHROMOSOME

BY SOMATIC CELL GENETICS

FLORENCE C. RICCIUTI AND FRANK H. RUDDLE

Department of Biology, Yale University, New Haven, Connecticut 06520

Manuscript received February 7, 1973 Transmitted by ERNEST H. Y. CHU

ABSTRACT

The intrachromosomal localization of three X-linked gene loci (PGK, HGPRT and GGPD) has been determined using a somatic cell genetic ap- proach. A human cell line possessing an X/14 translocation was used as one parent in the formation of human/mouse hybrids. The translocation separates the human X into two parts: Xp and t(Xql4q). The data indicate that all three X-linked loci segregate with the t(Xql4q) rearrangement product thus permitting their assignment to the X chromosome's long arm. Secondary rearrangements and data from other laboratories suggest that the order of the the three markers from the centromere to the distal end of the X long arm is PGK, HGPRT, GGPD. It was also observed that NP, an autosomal locus, segregated with the t(Xql4q) chromosome. This provides strong support for the assignment of NP to 14.

OMATIC cell genetics has proven to be an effective technique for the genetic analysis of mammals, especially man. During the past several years, using

human/mouse somatic cell hybrids, more than 26 human autosomal gene loci have been mapped to 15 chromosomes ( RUDDLE 1973).

Membrane fusion can be used to combine genetically different nuclei such as human and mouse into a single cell, Cells have been fused spontaneously (BAR- SKY, SORIEUL and CORNEFERT 1961), by inactivated Sendai virus (HARRIS and WATKINS 1965) or by chemical treatment (CROCE et aZ 1971). Human/mouse hybrids are particularly favorable objects for genetic analysis, because they preferentially segregate human chromosomes (WEISS and GREEN 1967). The mouse and human forms of homologous enzymes frequently have different electrophoretic mobilities. and can thus be distinguished. In addition, the mouse and human chromosomes can be identified by the application of recently-developed cytological techniques.

Linked relationships can be inferred by concordant or discordant segregation of human markers. If positive correlation exists between two human phenotypes, it can be presumed that the genes are syntenic; and similarly, positive correla- tions between human phenotypes and chromosomes provides strong support for the assignment of gene loci for particular phenotypes to particular chromosomes.

The general strategy of genetic linkage analysis in somatic cell hybrids, therefore, provides evidence for assignment of genes to chromosomes, but does not Genetics 74: 661-678 August, 1973

662 F. C. RICCIUTI A N D F. H. RUDDLE

provide information with regard to localization of gene loci to subregions of chromosomes. This finer gene mapping can be accomplished in two ways. HumanJmouse somatic cell hybrids can be examined for the appearance of al- tered chromosomes which have resulted from spontaneous chromosome breakage within a hybrid clone. The alterations could involve partial or complete deletions of either long or short arms or rearrangements, such as translocations. Such rearrangements have been observed in hybrid cells, and used to fix the location of the human thymidine kinase gene on the long arm of chromosome 17 (BOONE, CHEN and RUDDLE 1972). However, the chance of observing such rearranged chromosomes which occur sporadically in hybrid cells is small.

In addition to the studies just mentioned, it is possible to establish order and intrachromosomal relationships by using human parental cell lines in which a naturally-occurring stable chromosomal rearrangement has occurred. Such modi- fications of the chromosome essentially disrupt normal syntenic relationships and separate the chromosome into segments onto which gene loci can be mapped. In the work reported here, a human cell line (KOP) which possesses a naturally- occurring X/14 translocation has been used to study the intrachromosomal linkage relationships of the X-linked genes encoding hypoxanthine-guanine phospho- ribosyltransferase (HGPRT) , glucose &phosphate dehydrogenase (GGPD) and phosphoglycerate kinase (PGK). In the KOP cell lines, the XJ14 translocation has resulted in a physical separation of the long arm from the short arm of the X chromosome. Using the hybridization technique with the KOP cell line and employing HAT medium, which selects for the gene for HGPRT (LITTLEFIELD 1966), it should be possible to assign the X-linked genes for HGPRT, GGPD and PGK to specific regions of the X chromosomes, e.g., to either the long or short arm.

If the three X-linked gene loci encoding HGPRT, GGPD and PGK are located on both of the rearrangement products of the X chromosome, it can be assumed that they will segregate discordantly among the clones. If the three X-linked markers are all restricted to either the long or short arm, they will segregate concordantly in the clones. If an autosomal marker is located on the D-14 chromosome, it can be expected to segregate concordantly with any of the X-linked marker (s) to which it is translocated. Thus the experimental system serves not only as a means for assigning the X-linked genes but also for the assignment of autosomal markers to chromosome 14.

MATERIALS A N D METHODS

Cell h e r The human fibroblastic cell populations used in establishing hybrids were KOP-1 and KOP-2 cell lines derived from skin biopsies from a mother who is a translocation carrier and from her affected son, respectively. The translocation was first identified in the son who is a mentally retarded male with Klinefelter’s syndrome (J. M. OPITZ and P. D. PALLISTER, personal communication; SINISCALCO 1970). Examination of the chromosomal complement of the mother (KOP-1) revealed 46 chromosomes with an unusually long acrocentric and an additional small submetracentric chromosome. The morphology of the latter was similar to chromosomes of the E group. Autoradiographic studies of the chromosomal replication patterns showed that the X and D-14 chromosomes were involved in the translocation. The karyotype of this individual

CHROMOSOME LOCATION O F HUMAN GENES 663

I

FicunE 1 .--Ideogriim of KOP-2 (the son). Thc: chromnsonirs h a w t ) w r i stiiirwd w i t h quinacrine mustard. KOP-2 cells possess 47 chromosomes including five D group, two t(l4qXq) and one Xp chromosome.

represented a balanced translocation with one normal X and five D group chromosomes. Tritiated thymidine labeling studies also indicated that the structurally unmodified X chromosome repli- cated later in the cell cycle and is therefore presumed to be physiologically inactive.

The son from whom KOP-2 cell line (Figure 1) is derived possessed 47 chromosomes, including two t ( 14qXq) chromosomes presumably arising through meiotic nondisjunction. In addition, he possessed one Xp, one chromosome 14 and one Y chromosome. One of the t(14qXq) chromosomes was late laheling, as determined by tritiated thymidine incorporation followed by auto- radiography (J. M. O P I ~ , personal communication), and was assumed to be inactive.

The long acrocentric rearranged chromosome, designated t ( 14qXq), consists of almost a11 of chromosome 14 and the distal two-thirds of the long arm of the X chromosomr. The shorter submetacentric translocation product, Xp, appears to be composed of the short arm, centromrre and proximal third of the long arm of the X chromosome only. The t(l4qXq) chromosome is morphologically distinguishable from all others in the human genome. The Xp chromosome is indistinguishable from chromosomes 17 and 18 by arm ratio measuremrnts, but can be identified using various staining techniques to be described later.

We nrr indebtrd to DR. &nom KLINGER of the Albert Einstein School of Medicine, Bronx, New York, for providing us with KOP-1 and KOP-2 human cell lines. Fresh biopsy material from both KOP-1 and KOP-2 was also obtained with the help of Dns. J. M. OPITZ, University of Wisconsin, Madison, Wisconsin and PHILLIP PALLISTER, Boulder City, Montana. This material is now available from the Bank of Human Genetic Mutant Cell Cultures located at the Institute for Mrdiral Rcsrarch, Copewood Street, Camden, Nrw Jersey.

The Rag cell line, developed and described by K L m E (1970). was derived from a rrnal adenocarcinoma (malignant kidney tubule cell) carried in the BALB/cd strain of mice. The RAG cell line is rrsistant to 8-azaguanine due to a deficiency in hypoxanthinr-guanine phos-


phoribosyltransferase, which has been demonstrated electrophoretically and by activity measure- ments. RAG cells are unable to survive in HAT medium. RAG has a modal chromosome number of 74, including five metacentrics formed either by centric fusion of two acrocentrics or mis- division of the centromere resulting in the formation of isochromosomes (CHEN and RUDDLE 1971).

The KOP cell lines differ from the RAG cell line biochemically and cytologically. The human chromosomes can be distinguished from the mouse chromosomes using various staining techniques such as quinacrine mustard (CASPERSSON et al. 1970). Electrophoretic differences exist between human and mouse cell lines for the enzymes GGPD, HGPRT and PGK which are visu- alized by starch gel electrophoresis, as well as for 16 autosomally-linked enzymes.

The hybrid cell populations were maintained on Dulbecco Vogt Modified Eagle's medium (DVME) supplemented with 10% calf serum, and containing 100 units/ml penicillin, 100 pg/ml streptomycin and HAT mixture (DVMEBAT). The HAT mixture consists of l W M hypoxanthine, 4 x 10-7 M aminopterin, and 1.6 x 1G-5 M thymidine (LITTLEFIELD 1964).

Cells from KOP-1 and KOP-2 were fused with RAG cells using P-propiolactone inactivated Sendai virus according to a procedure previously described by KLEBE, CHEN and RUDDLE (1970).

50-10 c e l l s per T-25 f l a s k

I I s o l a t e d 6 subcl ones

TABLE 1

Protocol f o r subcloning experiments performed on primrry clone RK 3-5

RK 3-5

I

+RK 3-5za

+RK 3-5z j +RK 3-5zh'

+ denotes i n c l u s i o n i n coded s e r i e s o f s l i d e s

CHROMOSOME LOCATION OF HUMAN GENES 665

Hybrid cells were selected in DVME/HAT medium and isolated using steel cloning rings (PUCK, MARCUS and CIECIURA 1956). A total of 25 independent primary hybrid clones were isolated from the two hybridizations, 12 from KOP-2 x RAG and 13 from KOP-I x RAG. The hybrid clones were expanded and assayed biochemically and cytologically.

Isolation of Hybrid Subclones in Selective and Non-selective Media One primary clone, RK 3-5 was subcloned in nonselective medium DVME according to the

protocol presented in Table 1. Eight secondary clones were isolated, expanded and assayed biochemically. Two primary clones, RK 2-7 and RK 3-5 were subcloned in DVME/HAT selective medium following the protocol listed for RK 3-5 in Table 1 . Twenty-seven subclones were isolated and analyzed in the sams way as that described above. The same two primary clones were also subcloned in DVME medium containing 20 pg/ml 8-azaguanine (DVME/8-AZ). A total of 31 subclones were isolated and expanded in DVME/8-AZ selective medium.

Biochemical and Cytological Analyses All 25 primary and 66 secondary clones were assayed electrophoretically on starch gel for the

human forms of GGPD (RUDDLE, SHOWS and RODERICK 1968) and PGK (MEERA KHAN et al. 1971). Human activity for HGPRT was inferred by the method of drug selection using HAT medium and, in some instances, by direct electrophoretic procedure using a starch gel adaptation of the method of TISCHFIELD, BERNHARD and RUDDLE (1972). The clones were also assayed for the human forms of the following 16 enzyme phenotypes whose genes are autosomally linked: adenosine deaminase (ADA; E.C. 3.5.4.4) ( RUDDLE and NICHOLS 1971), glucosephosphate isomerase (GPI; 5.3.1.9) (RUDDLE and NICHOLS 1971), glutamate oxaloacetate transaminase (GOT; 2.6.1.1.1) (RUDDLE and NICHOLS 1971), indole phenol oxidase A and B (IPO A and B) (BREWER 1967), ismitrate dehydrogenase (IDH, 1.1.1.42) (RUDDLE and NICHOLS 1971), lactate dehydrogenase A and I3 (LDH A and B; 1.1.1.27) (RUDDLE and NICHOLS 1971), NADP-malate oxidoreductase (MOD; 1.1.1.37) (RUDDLE and NICHOLS 1971), NAD-malate oxidoreductase (MOR; 1.1.1.40) (RUDDLE and NICHOLS 1971), mannose phosphate isomerase (MPI; 5.3.1.8) (NICHOLS, CHAPMAN and RUDDLE 1972), nucleoside phosphorylase (NP; 2.4.2.1) (EDWARDS, HOPKINSON and HARRIS 1971), peptidase A, B and C (Pep A, B and C; 3.4.3.-) (RUDDLE and NICHOLS 1971), and phosphoglucomutase I (PGM I ; 2.7.5.1) (RUDDLE and NICHOLS 1971).

The electrophoretic assay for N P is a modification of the procedure described by EDWARDS, HOPKINSON and HARRIS (1971). A tris-ethylene-diaminetetraacetate (EDTA) -borate (TEB) buffer at pH 8.6 was employed instead of tris-citrate-borate lithium hydroxide buffer system at pH 7.2 used by EDWARDS et al. The qualitative assay for HGPRT was adapted to starch gel by E. NICHOLS and is based on the method of TISCHFIELD, BERNHARD and RUDDLE (1972). The starch gel is made using a citrate phosphate buffer at p H 6.8 with 35 mg DTT (dithrothreitol). The gel is loaded and run at 150 volts for 15 hours at 4°C. The cut surface of the gel is exposed to the reaction mixture consisting of 2.4 mg 5-phosphoribosyl-I-pyrophosphate magnesium salt (PRPP), 0.4 mg MgCl,, 20 ml 0.1 M Tris-HC1 pH 7.4 and 35 pl 14C-hypoxanthine for one hour at 37°C. The reaction mixture is then poured off, and the gel is placed in 0.1 M lanthanum chloride in 0.1 M Tris-HC1 at pH 7.0 for six hours at 4°C. The lanthanum chloride precipitates inosinic acid within the matrix. The gel is washed overnight to remove unreacted labeled hypoxanthine, dried, then exposed to X-ray film for seven days. The site of enzyme activity appears as an exposed spot on the film.

Because hybrid cells undergo progressive slight changes in chromosome constitution, it is important that the chromosome and enzyme preparations be made within 1-2 cell generations of the phenotype assays. Chromosome preparations were made on all primary and secondary clones according to the procedure described by MOORHEAD et al. (1960). Unstained slides from primary and secondary clones were scanned on a Zeiss light microscope with a 1 6 x or 2 5 x objective, using phase contrast. Metaphase spreads in which chromosomes were condensed, refractile or clumped were eliminated from the analysis. The slides were stained with 0.1% quinacrine mustard (QM) solution for 5-10 minutes, rinsed in running tap water for 5 minutes, mounted in citrate-phosphate buffer at pH 5.5 and the coverslip sealed with Kronig cement.

666 F. C. RICCIUTI AND F. H. RUDDLE

After QM staining the previously identified metaphases were located using a Zeiss dark field ultracondenser (N.A. 1.2-1.4), then photographed through a 1 0 0 ~ planachromat oil objective with adjustable diaphragm (N.A. 0.8-1.3) and an 8x ocular. All chromosomal analysis was done on prints or karyotypes, as opposed to scoring the cells by direct visualization at the microscope.

After staining with QM the cells were treated with a solution of 3 parts methano1:l part glacial acetic acid and then restained according to one of the following two procedures: (1) In some cases the slides were stained with 1.5% aceto-orcein for 5-10 minutes; or, ( 2 ) Slides were treated with 95% formamide at 67°C for 10 minutes (DEV et al. 1972), then stained with Giemsa in citrate-phosphate buffer at pH 6.8 for 20-30 minutes to reveal constitutive heterochromatin. The same metaphase spreads that were observed with QM were relocated and photographed a second time. This double staining procedure allows for more accurate identification of human chromosomes than can be made using either staining treatment alone.

It is possible that prior knowledge of the enzyme phenotype expressed by the hybrid clones might influence the identification of the human chromosomes of individual clones. To eliminate this bias, 36 slides from 10 selected subclones which were isolated in DVME/HAT or DVME/8- AZ medium and one of the two primary clones from which the subclones were derived were coded by a person uninvolved in the biochemical and cytological analyses. All human chromosomes including the rearranged human chromosomes were analyzed in the hybrid clones.

RESULTS

Enzyme Studies

Two fusion experiments were performed: KOP-1 x RAG and KOP-2 X RAG. The HGPRT deficiency in the RAG cell line and the presence of the gene for HGPRT in the human cell lines KOP-1 and KOP-2 permit the selection of humanJmouse hybrids by growing them in DVME/HAT medium. From the first hybridization (KOP-1 x RAG) 13 primary hybrid clones designated RK 3- were isolated in D V M E P A T medium. From the second hybridization (KOP-2 X RAG) 12 hybrid clones, termed RK 2- were isolated. These 25 independent primary hybrid clones were expanded and assayed cytologically and biochemically. The data derived from the two experiments were not significantly different, and the results were therefore combined.

The hybrid clones were primarily analyzed for the expression of the human forms of the three X-linked genes. Pedigree analyses have shown that the genes for HGPRT, G6PD and PGK are X-linked in man (SEEGMILLER, ROSENBLOOM and KELLEY 1967; DAVIDSOIS, NITOWSKY and CHILDS 1963; and VALENTINE et al. 1971). Somatic cell genetic analyses have confirmed these linkages (NABHOLZ, MIGGIANO and BODMER 1969; BOONE and RUDDLE 1969; GRZESCHIK et al. 1972; RICCIUTI and RUDDLE 1973).

Survival and growth of the RK hybrids in DVMEJHAT medium is dependent on the presence of the HGPRT. Thus, by inference, if the hybrid clones grow in DVME/HAT, then the human gene for HGPRT is present. In 25 hybrid clones, the human enzyme was present as determined by growth in DVMEJHAT and/or by direct detection of human HGPRT by starch gel electrophoresis (Figure 2). The electrophoretic mobilities of G6PD on starch gel are different for the mouse and human forms. All 25 primary clones showed both mouse and human forms and the heteropolymeric form which is composed of mouse and human subunits

CHROMOSOME LOCATION OF H U M A N GENES 667

Hypoxanthine- Guanine

Phosphoribosy It ransferase

+

HUMAN

MOUSE

ORIGIN 1 2 3 4 5 6 7 %

FIGURE 2.-Expression of HGPRT in hybrid clones. The enzyme is present in those clones isolated in DVME/HAT medium and absent in clones isolated in DVME/8-AZ medium. Because the mouse cell line, RAG, has no HGPRT activity, the mouse cell line LM(TK-) is also included. Channel 1 . . . . . RK 2-7, primary clone (DVME/HAT)

2. . . . .LM(TK-), mouse control 3 . . . . . KOP-2, human control 4. . . . . RAG, mouse control 5 . . . . . RK 3-520, subclone (DVME/8-AZ) 6. . . . . RK 3-5, primary clone (DVME/HAT) 7. . . . . RK 2-7zv, subclone (DVME/8-AZ) 8 . . . , . R K 3-51, subclone (DVME/HAT)

(Figure 3). Mouse and human forms of phosphoglycerate kinase (PGK) can also be distinguished from each other on starch gel. In 24 of 25 primary clones, the murine and human forms were observed (Figure 4). In one primary clone, RK 3 4 , derived from the fusion of KOP-1 x RAG, the human form of the enzyme was absent.

In the process of analyzing the primary clones for the presence of human forms of enzymes whose genes are autosomally linked, it was observed that nucleoside phosphorylase (NP) segregated as an X-linked marker. Nucleoside phosphorylase has been shown by EDWARDS, HOPKINSON and HARRIS (1971) to have a trimeric subunit structure and to be autosomally linked. Only 21 of 25 primary clones were assayed for the human form of NP, because of an accidental loss of four clones prior to the ascertainment of NP. In 20 of 21 clones the human homopolymer, two hybrid heteropolymers and the mouse homopolymer enzymes were observed (Figure 5 ) . In the one exceptional clone, RK 3 4 , the human form of the enzyme was not present, nor were the two heteropolymers. This same clone also showed discordancy among the X-linked phenotypes, having HGPRT and G6PD activities, but not PGK activity. The 20 clones which were positive for the human form of N P were also positive for the human forms of HGPRT,


Glucose -6- Phosphate Dehydrogenase

w - 7 - + MOUSE

HETEROPOLYMER HUMAN

-ORIGIN - k - 1 2 3 4 5 6 7

FIGURE 3.-Glucose-6-phospl1ate dehydrogenase phenotype of hybrid cells. The hybrid clones isolated in DVME/HAT medium possess both human and murine parental forms and the hctcropolymer. The subclonrs isolated in DVME/S-AZ medium have none of the human forms. Channcl 1 KOP-2, human control

2 RAG, mouse control 3 RK 2-7zr, subclone (DVME/8-AZ) 4 . RK 3-5za, subclone (DVME/8-AZ) 5 6 RK 3-51, subclone (DVME/HAT) 7

RK 3-5, Primary clone (DVMEWAT)

RK 3-6, primary clone (DVME/HAT)

GGPD, and PGK. The segregation of NF with the X-linked markcrs was strik- ingly significant when compared with the generally random segregation of 15 other autosomal human enzyme phenotypes among the hybrid clones.

In order to test further whether or not the concordant segregation was due to syntmy, two primary clones were subcloned in different selective media (Table 1). Sixty-six subclones isolated in the different media (DVME/%Az, DVME/ HAT and DVME) were studied for the expression of the four phenotypic traits. The results are presented in Tablc 2. The seltction pressure was directed at the gene for HGPRT. It had been shown by NARHOLZ, MIGGIANO and BODMER (1969) that growing cells in meclium containing 8-azaguanine enriched for cells lacking HGPRT activity. They further showed that the loss of enzyme activity

I

+

- [

CHROMOSOME LOCATION OF H U M A N GENES

Phosphoglycerate Kinase 6G9

MOUSE

HUMAN

-ORIGIN

1 2 3 4 5 6 7 8 9 FIGURE 4.-Electrophoretic pattern of phosphoglycerate kinase in hyhrid clones. In hybrids

isolated in DVME/HAT medium both mouse and human forms are present. In subclones isolated in DVME/8-AZ medium only the mouse form is present. The one exceptional subclone, m - 7 z v , is described in the text. Channel 1 . . . . RK 2-7zr, subclone (DVME/8-AZ)

2 . . . . .RK 2-7zv, subclone (DVME/S-AZ) 3 . . . . .RK2-7l,subclone (DVME/HAT) 4. . . . RK 2-7zt, subclone (DVME/8-AZ) 5 . . . . RK 2-720, subclone (DVME/S-AZ) 6 .RI( 2-7u, subclone (DVME/HAT) 7 . . . . . RK 3-5, primary clone (DVME/HAT) 8 . . .RAG, mouse control 9 . KOP-2, human control

TABLE 2

Srgrrgation data of X-IiriA-rd markrrs, GhPD, HGPRT a d PGK with autosomal marker, N P in primrrry clones and subclones isolntrd in differrnt srlectiue m d i a ( X markers/NP)

-~ . .__ . I'rmnry i Iwm +/+ -/- +/--/+ Discrepant clones -___- --______ RAG x KOP-1 13 0 1' 0 1 ' GGPD (+), HGPRT (+), PGK (-), N P (-) RAG x KOP-3 1 2 0 0 0 Subclones-

DVME/8-AZ 0 28 1 2." 1 GGPD (-), HGPRT (-), PGK (+), NP (+) 2" G6PD (-), HGPRT (-), PGK (-), NP (+)

Subclones-

Subclones- DVME/HAT 2G 0 1' 0 1'GGPD (+),HGPRT (+),PGK (-),NP (-)

DVME 8 0 0 0 58 28 3 3

6 70 F. C. RICCIUTI A N D F. H. RUDDLE

Nucleoside Phosphorylase

+

HETEROPOLYMER

HUMAN

- ORIGIN 1 2 3 4 5 6 7 8 9

FIGURE 5.-Electrophoretic pattern of nucleoside phosphorylase, a trimeric enzyme in structure. The segregation pattern of this enzyme was the same as that for the X-linked genes. Enzyme activity was present in clones isolated in DVME/HAT medium and absent in subclones isolated in DVME/B-AZ medium. The exceptional clone, RK 2-7zv, is described in the text. Channel 1. . . . . RAG, mouse control

2 . . . . . RK 3-5, primary clone (DVME/HAT) 3 . . . . . RK 2-7zu, subclone (DVME/B-AZ) 4. . . . . RK 2-720, subclone (DVME/B-AZ) 5 . . . . . RK 2-7zt, subclone (DVME/%AZ) 6 . . . . .RK2-71, subclone (DVME/HAT) 7. . . . . RK 2-7zv, subclone (DVME/8-AZ) 8. . . . . KOP-2, human control 9. . . . .RAG, mouse control

was due to segregation of the human X chromosome rather than to a mutational event. Growth in DVME/8-AZ medium favors those hybrid cells which have lost the gene for HGPRT. Although the selection is directed solely at the gene for HGPRT, the other two X-linked markers, GGPD and PGK, as well as NP will segregate depending on their syntenic relationship to HGPRT. Any gene located on the same rearranged chromosome as the gene for HGPRT would segregate concordantly with HGPRT. Twenty-six of 27 independent subclones isolated in DVME/HAT medium retained the human genes for HGPRT, GGPD and NP. Only one subclone, RK 3-51, showed discordant segregation of the X-linked genes, retaining HGPRT and GGPD but lacking PGK and NP. Thirty-one subclones were isolated in DVME/8-AZ medium. Of these, 28 showed concordant loss of the X-linked genes and NP. In one exceptional clone. RK 2 - 7 ~ ~ ~ the human forms of PGK and N P were observed, but no human GGPD or HGPRT activity was detected on starch gel electrophoresis. Two additional subclones, RK

CHROMOSOME LOCATION OF H U M A N GENES 671

3-5za and RK 3-5zh’, showed concordant loss of the X-linked markers, but retained the human form of NP. The presence of the NP phenotype in the absence of the X-linked markers is probably due to the retention of the normal chromosome 14. However because the banding pattern of chromosome 14 closely re- sembles some of the murine chromosomes, this determination could not be made. Eight subclones, all derived from RK 3-5, were isolated in DVME medium. All four human phenotypes were present in these subclones.

It would appear that the concordant segregation of the X-linked markers in selective and nonselective media is due to linkage to a common chromosome. This evidence, taken together with the segregation of NP as an X-linked gene, would strongly support the assignment of the four genes to the long rearranged chromosome t (14qXq). This interpretation is most probably correct since nearly the whole chromosome 14 contributes to the long rearranged chromosome t (14qXq). The possibility, although improbable, still exists that the genes are distributed on both rearranged chromosomes which segregate concordantly for unknown reasons. Cytological analyses on primary and secondary clones have served to support the interpretations given above and to decide among the alternative possibilities.

Chromosome Studies

The rearranged chromosome(s) on which the X-linked gene loci and NP are located will segregate concordantly with the expression of the human phenotypes. If the four genes are located on one or the other rearranged segments, then only one of the rearranged chromosomes should be uniformly present. If the genes are distributed on both rearranged X-pieces, then both chromosomes should be observed when all four markers are present.

Sixteen of the 25 primary clones were analyzed cytologically for the presence of the long rearranged chromosome t (14qXq) and for the shorter submetacentric chromosome Xp. The results of chromosomal analysis of 16 primary clones for the X/14 translocation products are presented in Table 3. In 15 of 16 clones, the iong rearranged chromosome t (14qXq) was present. The Xp chromosome was observed in only two of the 16 primary clones, RK 2-7 and RK 3-5, with any appreciable frequency. In one exceptional clone, RK 3-4, which was negative for the expression of PGK and Np but positive for HGPRT and GGPD, the long t ( 14qXq) chroniosome was absent. In this clone a small submetacentric chromosome similar in arm ratio to the Xp chromosome was identified in two of 33 cells. However, i t did not have the characteristic sharp band as revealed by quinacrine mustard in the middle of the short arm.

Two primary clones, RK 2-7 and RK 3-5, which were subcloned did possess a small submetacentric chromosome. In RK 2-7 a small chromosome (E?) similar in arm ratio to chromosome 17 and the Xp Chromosome was observed. This chromosome did not have the distinctive banding pattern which was characteristic of the Xp chromosome, nor did it resemble the banding patterns of chromosomes 17 or 18. The t (14qXq) chromosome was present in the great majority of cells. In RK 3-5 a definitive Xp chromosome was identified by its distinct band-

6 72 F. C. RICCIUTI AND F. H. RUDDLE

TABLE 3

Cytological analyses of X / 1 4 translocation products in primary clones

Primary clone

2-1 2-2 2-6A 2-6B 2-7 2-10 2-14 2-15

3-1 3-2B 3-4 3-5 3-6 3-8 3-12 3-14

Chromosome range

(105-133)

(126-171)

( 1 07-1 3 8) (1s-158) (167-206) (107-1 26)

(130-176) (1 10-139) (117-145)

(1 14131 ) ( 92-146) (116-156) (1 05-1 60)

(107-156)

( 54-73 )

( 105-156)

Number mean

117.4 129 142.1 63.6

120.5 143.3 181.7 119.1

147.6 123.7 130.8 128.9 122.3 134 138.1 124.0

Total no. of cells

8 6

30 14 27 18 6

15

7 15 33 20 9

13 12 12

Number with

t(14qXq) Percent

7 87 4 66 w 77 11 78 19 70 14 78 4 66

13 86

4 57 12 80 0 0

17 85 6 66

10 80 10 83 10 83

Number with Xp Percent

0 0 0 0 l? 33 0 0

21(E?) 74 13 5? 0 0 0 0

0 0 1 6 2? 63 8 40 a 0 0 0 1 8 0 0

ing pattern as revealed by quinacrine mustard staining. The t (14qXq) chromosome was also present in most of the cells. In one primary clone, RK 3-8, a structurally unmodified human X chromosome was observed in four of 13 cells, but the Xp chromosome was absent. No other RK 3- primary clone was observed to contain a normal X chromosome. This finding indicates that the concordant segregation of the X-linked gene loci observed in the primary and secondary clones is due to the presence of the t (14qXq) chromosome and not due to the presence of the structurally-unmodified X chromosome.

From the cytological data it may be concluded that all four gene loci are located on the long rearranged chromosome, since this chromosome was identified in 15 of 16 clones. A positive correlation was not observed betweell the Xp chromosome and any of the four markers. Thus the possibility that any of the markers are located on the short chromosome can be ruled out.

It was also necessary to look for the X/14 rearranged chromosomes in the de- rivative subclones, particularly those isolated in DVME/8-AZ medium and the two subclones in which discordant segregation occurred between the X-linked markers and NP. Chromosomal analyses were performed on a coded series of slides from selected subclones. The cytological results are presented in Table 4. From the data it is apparent that the random segregation of the long rearranged chromosome is dependent on the selective medium employed. In those subclones isolated in DVMEJ8-AZ medium the t (14qXq) chromosome was never observed. In subclones isolated in DVME/HAT medium the long t (14qXq) chromosome was observed in all but one subclone, RK 3-51. This subclone was discordant in the segregation of the X-linked markers, having HGPRT and G6PD activities but not PGK or NP activity. Three oE six subclones of RK 3-5 isolated in DVMEJ8-

CHROMOSOME LOCATION OF H U M A N GENES 6 73

TABLE 4

Cytological analyses of X / 1 4 translocation products in subclones

Subclcne Isolation (RKI medium

Chromosome range

Number mean

Total no. of cells Percent

2-7ZV

2-7h 2-7j 2-7i L7zd 2-71 2-7zj 2-7zj

2-72s

3-5zj 3-5zh' 3-5za

2-7zg

3-51

DVME/8-AZ DVME/HAT DVME/HAT DVME/HAT DVME/8-AZ DVME/HAT DVME/8-AB DVME/8-AZ DVME/B-AZ DVME/8-AZ

DVME/8-AZ DVME/8-AZ DVME/8-AZ DVME/HAT

(1 03-132) ( 115-120)

( 105-140) (1 1f3-130)

(120-135) (121x135) (110-135) (114-132)

( 96144) ( 97-133)

(1 18-136)

( 98-125)

(138-140)

( 95-137)

124.2 117.6 114.4 121.2 123.8 138.6 125.9 125.9 123.9 122.9

44 8

21 33 15 3

27 27 31 23

0 7

17 24

0 0 0 0 0 0

0' 88 81 73 0 0 0 0 0 0

Number with

Xp or E? Percent

14(E?) 31 0 0 0 0 0 0 0 0 I(E?) 33

14(E)? 66 14(E)? 66 15(E?) 50 7(E?) 31

123.2 17 0 0 0 0 115 8 0 0 ~ ( X P ) 88 124.3 24 0 0 15(Xp) 62 125.6 19 0 0 0 0

AZ medium were analyzed in the coded series of slides. Two of these contained an identifiable Xp chromosome, as did the primary clone from which the subclones were derived.

Three subclones, RK 2 - 7 ~ ~ ~ RK 3-51 and RK 3-Sza, were especially interest- ing phenotypically because they were the only three subclones which showed discordancy among the four markers. They showed either separation of NP and PGK from HGPRT and GGPD or they lacked the three X-linked markers. Cyto- logical studies are described below.

RK 2-7zv: In this subclone, the enzyme phenotypes were HGPRT (-) , G6PD (-), PGK (+) and NP (+). Forty-four cells were cytologically analyzed and the t (14qXq) chromosome was never observed. The Xp chromosome was not observed. However, the E? chromosome was present in 14 of 44 cells.

RK 3-51: There was discordant segregation of the X-linked markers and NP in this subclone derived from RK 3-5. The enzyme phenotypes of the clone were IIGPRT (I-), G6PD (+), PGK (-) and NP (-). Neither the t(l4qXq) nor Xp chromosome was present. A new chromosome was identified which was unlike any other human or mouse chromosome. Multiple copies of this chromosome were found in every cell, the range being from two to five per cell. This chromosome could be the result of rearrangements among human or between human and mouse chromosomes. Since this subclone showed separation of X-linked genes, quite possible that part of the X chromosome carrying the genes for HGPRT and G6PD may be part of this chromosome. The resolution of the cytological techniques was insufficient to distinguish between the various possibilities.

RK 3-5za: (Figure 6) The enzyme phenotypes exhibited by this subclone were HGPRT (-) , GGPD (-), PGK (-) and NP (+) . The t (14qXq) chromosome was not present. but the Xp chromosome was present. The normal human chro-

6 74 F. C. RICCIUTI A N D F. H. RUDDLE

~ ’ I G U I ~ F . G,-Irlrogri~ii~ c i f RK L5zz1, i i suhclonr isolntrtl in T~V\ l l~~ /8 - . \ 7 , inedium. The enzyme phenotype is: HGPIIT ( - ), (XPI) (-), P G K (-) and N P (+ ) . The long t(l4qXq) chrrimo- some was never observed, but the short Xp chromosome was present in 62% of the cells. The expression of the human form of NP is probably due to the presence of the normal chromosome 14 wtiich cannot be irlentified from murine chromosomes with similar banding patterns.

mosome I 4 was not identifiable because of similarity of its banding pattern to scveral murine chromosomes.

The results from the cytological analyses of the subclones provide additional support for the assignment of the four markers to the t (14qXq) chromosome. In the thrce suhclones with discordant segregation of the markers. the t( 14qXq) chromosome was never observed. I t can be postulated that a secondary rrarrange- ment has occurred in the t (14qXq) chromosome resulting in subsequent translocation to either mouse or human chromosomes. The cytological results. together with the biochemical findings from RK 3-5za, argue strongly against any of the four markers being on the Xp chromosome. The Chromosome was present though the X-linked gene loci were absent. In addition, mechanisms preventing segregation between the rearranged chromosomes can be dismissed.

DISCUSSION

Somatic cell hybridization has been used in this work to study the intrachromo- soma1 linkage relationships of the three X-linked gene loci for HGPRT, GGPD

CHROMOSOME LOCATION O F HUMAN GENES 675

and PGK. The striking concordant segregation of the X-linked markers in 88 of 91 primary and secondary clones and the finding that the autosomally-linked gene for NP segregated concordantly with the X-linked genes indicate that the four gefie loci are located on the t ( 14qXq) rearranged chromosome. Concordant segregation of the long t(l4qXq) chromosome and the four markers was the rule. When there was separation of the X linked genes, the long t (14qXq) chromosome was absent.

Segregation data from the RK series of hybrids in which separation of the X-linked markers occurred would indicate that the gene for PGK is more proximal to the gene for N P than those of HGPRT and GGPD in the t (14qXq) chromosome. Given the nature of the X/14 translocation, the apparent relationship of the above separation data to the actual physical location of the three X-linked genes on a structurally-unmodified X chromosome would place the gene locus for PGK closer to the centromere than either of the other two genes. Two possibilities exist for the order of the remaining two genes, HGPRT and GGPD on the X chromosome:

1) centromere.. . PGK . . . HGPRT . . . GGPD 2) centromere.. . PGK. . . GGPD.. . HGPRT The isolation and growth of the hybrid clones and subclones in the various

selective media was dependent OP either the presence or the absence of the human gene for HGPRT. Strong selective pressures were exerted on this gene. In the first linkage model, if a single break occurs between PGK and HGPRT, then we would expect to see HGPRT and GGPD segregating together and apart from PGK. In the secoEd linkage model, if a break occurs betw-en GGPD and HGPRT, we would expect to see separation of HGPRT from GGPD and PGK. In three instances separation occurred between PGK and HGPRT. This would support the first model, which considers the order of the genes as: centromere . . . PGK . . . HGPRT . . . GGPD.

Independent supporting evidence for our orderipg of the three gene loci comes from a series of human/mouse somatic cell hybrids formed from the fusion of RAG cells with cells from a human cell line possessing an X/19 translocation (P. GERALD, personal communication). In this humac cell line, one of the rearranged chromosomes consists of the short arm, centromere and proximal half of the long arm of chromosome 19 and the distal half of the long arm of the X chromosome. The other reciprocally-arranged chromosome of this translocation consists of the short arm, centromere and proximal half of the long arm of the X chromosome and the distal half of the long arm of chromosome 19. The breakpoint in the long arm of the X chromosome which resulted in the X/19 translocation is distal to the characteristic bright band which is revealed by quinacrine mustard staining. The breakpoint in the long arm of the X chromosome which resulted in the KOP XJ14 translocation is proximal to the bright band and thus to the breakpoint which lead to the X/19 translocation. Preliminary biochemical data from a series of hybrids have revealed that human forms of GGPD, HGPRT and GPI (assigned to 19, MCMORRIS et al. 1973) are expressed, but human activity for PGK is not. No cytological analyses have been performed as yet.

676 F. C. RICCIUTI AND F. H. RUDDLE

The data taken together from the two hybrid series would indicate that the genes for HGPRT are on the distal half of the long arm of the X chromosome and that the gene for PGK is on the proximal half. In addition, the gene for PGK is located in that part of the X chromosome which is between the two breakpoints which lead to the XJ14 and X/19 translocations, respectively.

Additional supporting evidence comes from a series of human/Chinese hamster hybrids in which a human cell line possessing an XJ3 translocation was used as oce parent (D. BOOTSMA, personal communication). In this translocation the breakpoint in the X chromosome is at the most distal part of the long arm. The breakpoint in chromosome 3 is in the proximal half of the long arm. The distal half of chromosome 3 has been translocated to the X chromosome and the extreme distal part of the X chromosome has been translocated to the proximal long arm of chromosome 3. Preliminary biochemical and cytological segregation data in this series of human Chinese hamster hybrids indicate that there is con- cordancy between the presence of human forms for PGK and GGPD and both rearranged chromosomes, respectively. The expression of GGPD could be cor- related with the translocation product containing the extreme distal segment of the X long arm. Thus, the gene for GGPD can be placed tentatively in the distal part of the long arm of the X chromosome.

Another study employing the same KOP X/14 translocation has been reported by GRZESCHIK et al. (1972). Their data, collected from a series of KOP-1 X mouse and KOP-1 X hamster somatic cell hybrids, to a large degree agree with those reported here showing preponderant concordant segregation of the three X-linked markers. In their study of the clones in which separation occurred among the X-linked genes, the separation was always between PGK and HGPRT. A close correlation between the presence of the t (14qXq) chromosome and human PGK was noted, but not between the t (14qXq) chromosome and the other two gene loci when they were separated from PGK. When only G6PD and HGPRT were present, the t(l4qXq) chromosome was absent. When all three genes were present or absent depending on the selective media used to isolate the subclones, the long t (14qXq) chromosome was present or absent, respectively. The authors have interpreted their results to support the idea that the gene for PGK is on the long arm of the X chromosome and the genes for HGPRT and GGPD are on the short arm, although the Xp chromosome could not be identified in hybrid cells in which these two genes are expressed. In addition, to account for the preponderance of clones and subclones showing concordant segregation of the three X-linked markers, the authors postulate that some mechanism is op- erating to cause retention of both translocated chromosome products or segregation of both. The data obtained by our study militates against this interpretation.

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