(Receivetl August 19th, 1963). lNTRODUCTION. XCEPTJONALLY large human Y chromosomes were recently de-. E scribed in 15 male members of 5 families ...
E L O N G A T I O N AS T H E P O S S I B L E M E C H A N I S M OF O R I G I N OF L A R G E HUMAN Y C H R O M O S O M E S AN AUTORADIOGRAPHIC STUDY By J O H A N IVEiVNSTROiM and ALBERT D E L.4 CHAPELLE THE F O L K I I ~ L S A N INSTITUTE FOR GENETICS, T H E MlNERVA FOUNDATION INSTITUTE FOR
blEDlCAL RESEARCH, A N D T H E FOURTH X1EI)ICAL UNIVERSITY CLINIC, HELSINGI~ORS,FINLANI)
(Receivetl August 19th, 1963)
lNTRODUCTION
E
XCEPTJONALLY large human Y chromosomes were recently described in 15 male members of 5 families (DE LA CHAPELLEet al., 1963). It was shown that the feature probably has no influence on the phenotype of the carriers of the trait or their offspring, as had been postulated earlier by BISHOPet crl. (1962). Since large Y chromosomes :ire rather coninion among normil subjects, it was considered of interest to investigate whether they reflect altered chromosomal behaviour (elongation) or different chromatin content (e.g. duplication or translocation). To evduate this question, a new tool in human cytogenetics, namely autoradiography of chromosomes labelled with tritiated thymidine (LIMA-IIE-FARIA et crl., 1961), was employed. ]\Then tritiated thymidine is added to cell cultures, it becomes incorporated into the nuclei during interphase. At the subsequent metaphase, the isotope can be recognized in the chromosomes with the aid of autoradiography. It was shown by LIMA-DE-FARIA et al. (1961) and GILBERTet al. (1962) that in this way a11 the human chromosomes, including the Y, become labelled. I n males, grain counts revealed a fairly even distribution of the isotope over all the chromosomes, :ilthough some differences were encountered. We postulaled that if the abnormally large Y chromosome conl:iins more DNA than the normal Y chromosome, it should incorporate more tritiated thymidine than a normal Y. Three subjects with large Y chroniosonies and three normal male control subjects were investigated, and
346
.IOI-lAN WENNSTHGM A N D ALBEHT IIE L A Cl-lA1'ELLE
the ratio of grain counts on tlie '1' was found lo be the groups.
s;iiiic
in the two
Material and methods Three male subjects with large Y clironio~oiiie~ were used. Two of Iheiii hiid hypog01iadi51ii (propositi A aiid B of IIE LA CHAPELLEet ( [ I . , 1963), and one was the llrother of ;I third propositus (11). The three control subjecls were clinically atid cytogenelically normal. Leukocyle cultures were p r e p r e d according to HUNGEHFORII ef rtl. (1959). After about 72 hours' culture, tritinled thymidine, :ind one hour later, colchicine, were Lidded. After 3-4 hours, harvesting was begun and slides prepared. A coating of photographic emulsion W:IS applied with the aid of a g h s s rod (LEHMANN, 1962). After about (5-8 hours' exposure, the slides were processed, stained with Giemsa'5 stain, :ind iiioiiii t ed. r , 1 he preparations were screened for good mitotic cells with appropriate 1:ibelling. For ;I cell to be \uilable for Ihis kind of investigatioii, certain criteria must be fulfilled, namely: ( 1 ) good spreading wilhout ovcr1:ippitig of tlie cliromosoiiic4, ( 2 ) good but not too intense staining of the chroiiio~oiiie~ to allow disti nc t i ot i be tween c h ro nioso m cs a nd gra i 11s i lid i ca 1 i t i g ra d ioac L i v i t y , ( 3 ) even distributioii of the photogr:iphic emulsion, and (4) absence of background activity interfering with the correct interpretation of the grain counts, :tiid of loo heavy I~ibelling,mukiiig c h r ~ i i i ~ s ~identification tne dil'ficult.
When these criteria are applied, the iiutnber of cells suil:rble for investigation is limited. Preferably, the chrotiiosoiiies of cells to be investigated should be annlysed in delail to prevent Inisinterpretation of individual chrotiio~omes.Thus, grain scores can be made of all individually recognizable chrotiiosoiiies and of groups of chromosomes. This requires preparations of exception:illy high qiiality. For tlie purpose ol' this investigation, however, i t w i \ only necessary to identil'y the Y and ascertain that the cell contained 46 chromosomes, since there W:IS no mosaicisni in the cases examined. Therefore, a coiiiplete analysis w:rs only done 011 a cerlnin number ol' cells, aiid in the rest the 1 ' chromosome w:is identified without any further analysis. The identification 01' the Y chroiiiosome was possible in most cells because ol' ils lieteropylciiotic appe:ir:itice. Occasionally, a n otherwise good cell had to be discarded because the 1' chromosome could not he recognized.
347
ORIGIN O F L A R G E HUMAN Y CHROMOSOMES
TAIJLE 1. Grciiii corriits on the Y chromosome and on the other 45 chromosomes registered .sepcircitel$j in 54 cells f r o m 3 subjects w i t h large Y chroniosonies nnd 41 cells from 3 subjects w i t h 11 or ni nl Y ch r o i n os onies. Sorinnl
2
1 0 1 4 1 0
24 73 50 63 64 95
2 0 1 1 0 1 2 3 2 1 1 0 1 0 1 0 1 O 0 0 0 0 2
88
56 26 29 39 84 71 121 45 53 110
92 30 1G
0 1 2 0 1 4 0 2
82 15 39 42 37 91 78
1
61 :I6 74 53 84 97 22 34 4 .5 65
1 1 0 1 2 2 0 1 1
Y chroniosonies 2
1
$18
58
177 99 85 56 48 42
1 1 0 1 0 0 2 0 1 2
0 5. 3 0 8 4 0 1 0
212 li0 180 55 121 179 110 33 103 101 47 21 208 151 82 167 99 21 62 43
39
2198
61 i5
4
3
0
20 43 4 33 28 28 62 119 41 12 42 15
10
583
1 0 1
2
1 0 2 2 0 2 1 1
0 0 0 1 0 4 1
($4 95 18 22 54 51 115
6
419
in 7 (i
G 12 23 5 8 31
Total: 26 1375
20
105G
348
JOHAW W E N N S T R 6 M A N D ALBERT DE LA CHAPELLE
T.4IJL13 2 . S u m s of qmin scores f o r the I’ m d the other 45 chromosomes from 3 subjects with large, cind 3 w i t h normal I‘ chromosoines.
I
1,nrge Y cliroiiiosoiiies
I1
Sol.lllnl
Y
CIll.OIlIOSOIII(~S
RESULTS I n every cell examined, the grain scores of the 1’ chromosome, and .of :ill the other 45 clironiosonies together, were recorded separately (Figs. 1-2). The scores are presented in Table 1. The sums of the grain score ratios of the six subjects :ire shown in Table 2. As can be seen, in 54 cells from 3 individuals with large Y chromosomes, the ratio of yrciins on Y I grains on the other 45 chromosomes was 5413016. In 41 cells from 3 subjects with normal Y chromosomes, the correspondiiig figures were 55/3200. Thus the large Y cliroiiiosoiiies labelled alniosl exactly ;is heavily as the normal 1’ chroniosomes. The data a r e too few, however, to permit statistical significance to be claimed. 13ad i a I i on-i nduced c h rom osoni e a11n or iii a1i ties , such a s dice n t r ics , fragments and different misdivisions, were frequently seen. This phenomenon w a s also noted by BENDEH et rrl. (t962).
DISCUSSION The assumption that a n abnormally large Y chro~iioso~iie \vould 1:ibel more intensely if it contailis more DNA than n iioriiial Y chromo\onie, may he criticized. It might be argued that since the large Y does not cause developmental disorder, its possible excess of chromatin would be “inactive” and be les5 heavily labelled than more “active” chromatin. Yet the “i iiac t i ve” sex -ch rom a t i 11-form i n g X chromosome labels 1a I er , :i nd t he ref o re more i 11 tensely , t h a 11 a n y o I her chromosome ( M OHI sti I M A et ( i l . , 1962; GILBERT et crl., 1962). Every chromosome must evidently incorporate thymidine to IN able to duplicate. By the autorndiogrnpliic method we therefore measure the time sequence of DNA synthesis only. I t appears legitimate to postulate that large and normal 1 ’ ‘ clironiosoines
1
Figs. 1-2.
13s:iinplcs o f mitotic cells I:il~clletl with tritintetl thyniidine. The large Y cliroiiiosoiiics :ire 1ii:irked by arrows. Note absence of b:ickgrountl radioactivity.
ORIGIN O F LARGE HUMAN Y CHROMOSOMES
349
synthesize new DNA at the sanie time. It seems possible at least, therefore, that our assumption is correct. Some sources of error are inherent in ;I study like the present. Occasional misinterpretation of the Y chroniosonie is possible, especially i n Ilic normal cases, in which the Y chroniosomes very closely rcscntble the smallest :tcrocentric autosomes. Radiation-induced chromosome abnormalilicr may interfere with identification. This is especially true when fragments similar in size to the 1 ' chroniosonie are present. 13y the strict application of the criteria listed above for tlie choice of cells to be esnmined, most errors may be avoided. Additional S chromosomes label late in tlie S period (ROWLEYet d., 1963). If, :IS was discussed by DE LA CHAPELLEet nl. (1963), the possiblc excess of chromatin of the large Y represented material tr:inslocated from an S chromosome, this segment would be expected to 1:tbel late. T ~ L Ithe S results of this study do not support the theory of translocation from a n X. The fact that large Y chromosomes label a s heavily as nornial Y chroniosotiies may be interpreted in many ways. I n our opinion, the most plausible conclusion is that the large Y only represents a n elongate of the DNA present in a normal 1'. Thus, the difference in size would be a mere morphological or behavioural peculiarity without functional significance or difference in m i s s . This is also supported by the fact that large Y chroniosonies seem to be :I rather coninion feature in the nori n a l population, and do not cause developmental disorder. Little is yet known about the time-sequence and functional details of DNA synthesis a s reflected by the incorporation of tritiated thymidine. It is therefore not possible to make a final assessment of the role and significance of large Y chroniosonies. ..lcl;noruletlgements. - \Ire wish to thank 1)r. OHLA L E H h l A N N for his kind help with the autoradiographic technique. This work w a s supported by grants from 'I'hc F i n s l u Liil~nresiillskapet,The Sigrid Juselius I'oundnlion and The hfcdical Commission 0 1 the Finnish State.
SUMMAHY l'ritiated thymidine was incorporated into leukocytes grown in vitro from 3 subjects with large Y chroniosoiiies and from 3 subjects with nornial Y clironiosonies. Autoradiography of mitotic cells showed that the large Y and the normal Y were equally labelled, as compared with the labelling of all the other 45 chromosomes. This indicates the possibility that large Y chroniosonics conlain Ihe sanie amount of DNA as
X50
JOIlAN WENNSTHOM AND ALBERT 1lE LA CHAPELLE
norm:rl Y chromosomes. T h e large Y would then represent only :I behavioural peculiarity (elongation) rather than a difference in DN.4 coiitent. Other interpretations are possible.
Literature cited ~ I E X D E IX ~I . , A,, Goocll. 1’. C. :ind PRESCOTT, 1). hI. 1962 Aberrations induced in 1iuni:rii leukocyte cliroiiiosoiiics 1)s *H-lal~clednucleositles. - Cytogenetics I: 65-74. IIisiioiI? A., I I L A X K , C. 1;. a n d HUWI’ER, 1-1. 1962. Heritable variation in the length o f the Y cIIrniiiosoiiic. - 1,:incet 19G2 ( 2 ) : 18-20. CIIAI~EILI:. r\. D E L A , I-IORTLING, II., I
