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S T U D I E S ON T H E S T I ~ U C T U I ~ E OF T H E CHI%Oh![OSOMEs

C H R O T O G O N U S I N C E R T U S ]~OLIVAI~ (ACI%.[]).[])AE) 0:~

BY M. D. L. SRIVASTAVA

Zoology Department, Allahabad U,nive~'sity, Allahabad, l',,di(l

(With Plates 19 and 20 and Sixteen Text-figures)

(Received 8 Aug~tst 1953)

INTRODUCTION

Two important questions concerning the morphology of chromosomes, wh ich are still far from satisfactorily settled, relate to the nature of chromomeres and the character of the so-called 'lamp-brush' processes. Most cytologists at present accept the chromomeres as definite granules occurring on the chromonemata (Darlington, 1937; White, 1945; Heitz, 1935; Huskins,. 1941; Kuwada, 1939; Koltzoff, 1938; etc.). There are ccrtaiu iuvestigatere, however, who claim to have resolved chromomeres into helices of a sph:al and according to whom the leptotene thread is already spirally twisted (Kaufmann, 1931 ; Smith, :[932; Koshy, 1934, 1937; Naithani, 1937; Swanson, 1943). Recently the chromosomes of male germ cells of a number of grasshoppers have been examined by I~is (i[945) from this point of view, who comes to the conclusion that the 'chromomeres do not exist as definite structm.es '. They are simply the 'misinterpretations of gyres of the chromonematic helix (leptotene, somatic prophase)'. The specifically paired chromomeres of zygotene chromo- seines he considers as ' the paranemic spiral of the paired bivalent'. The more recent work of ~r & l~{omma (1950) furnishes evidence to the same effect. They maintain that ' the leptotene clu'omosome consists of a uniform coiled thread and that what has be6n described as chromomeres are in reality gyres or twists in the chromoncmata'.

As regards the 'lamp-brush' fibres, Ris (1945) contends that they are 'the gyres of the major coils of the individual chromonemata, which have laterally separated from each other'. Hsu (1948) has put forth an ingenious hypothesis, linking up spiralization, hetero- pycnosis and formation of 'lamp-brush' processes. His ideas regarding the nature of 'lamp-brush' processes are incompatible with Ris's views, but have much in common with Goldschmidt's interpretation (Calvin, Kodani & Goldschmidt, 1940) of the 'lamp-brush' fibres, according to which, as a multiple division of chromatids into bundles of chromo- nemata takes place, some of the latter remain unstretched and form loops, appem'ing as 'lamp-brush' fibres. Serra (1947) has given out a similar interpretation. Duryee (193% [938, 1939, 1941), on the other hand, has shown that, in the oocytes of frog and sala- mander, lateral loops grow out from the chromomeres and are shc(l later on into the cytoplasm, h~ore recently (1950) he has reaffirmed his earlier conclusions. KoMo~ (1988) also held that they are side branches of chromomeres. Painter (1940) originally thollght that the 'lamp-brush chromosomes' result fi'om endomitosis and represent aggregated chromosomes, parts of which are given off ultimately into t;he cyt, oplasm. A year later (1941) he reached a different conclusion and thought the 'lamp-brttsh' fibres to be derived from the chromosome matrix.

M. D. L . S~rVASTAVA 481

~his in.vestigatiou was undertaken with the object of making a detailed report on the ~0mosome e~ of a common grasshopper and also to obtain and consider the bsari~g of this ma,terial on the questions mentioned above.

I ' V [ A T E I ~ I A L AND M E T H O D S

8pe0islens of the grasshopper, Ch~'otogonus ince~'tus Bolivar, were collected at Allahabad dzri~g ~he m.ont]ts May-September 1949. The testes were fixed in Bouin, Bouin-Allen, ~c1 corrosive subli mate-acetic. Sections were stained with Newton's gentian violet, with ~Ia~ll's methyl-l)lue eosin, and with basic fnchsin according to Feulgen's method. Aceto- carsliIle squash preparations were also studied.

0BSm~VATIO~S

~permalogoni(d d~;vision. Nineteen acrocentric chromosolnes appear at the spermato- 1r[I ~ -- p g01iial metaphase (. ex~-ng. 1). Neither at prophase nor at metaphase can ~]ie X-chromo-

some be differentiated from the autosomes, as it behaves like the latter in the degree of condensation and staining reaction, showing no heteropycnosis. At late prophase the

Text-fig. I. Text-fig. 2. Tsx~.iig. 1. Spermatogonia,l metaphase, showing nineteen acrocentric clu'omosomes. Boahl, N ann's methyl-

bhie eosin. • 2000. Tszl.fig. 2. Spermatogoni~d anaphase. :Boron, 1Kann's methyl-blue eosin. • 2000.

0b0mosomes attain the size characteristic of metaphase, but in the staining reaction there is a noticeable difference, the prophase chromosomes staining blue with ~ann ' s methyl-blue eosin, and the metaphase chromosomes being constantly eosinophil. Also there is some slight difference in volume, which might be due to a difference in the mount of matrix. At anaphase one pair of separated chromosomes lags behind somewhat (Text-fig. 2).

Mdosislpropha, se. The leptotene chromosomes are observed as finely granular and ~ghtly stMMng, slemler threads (Text-fig. 3). The ehromomeres are not found reducible b ~dsts of a spiral, and the threads appear consistently single and beaded. At zygotene the chromosome threads pair without forming a bouquet, and there is no synizesis (?ext-fig. 4). The paehytene threads are found to possess numerous lateral processes or 'lamp-brush, fibres, which stain like the mMn parts of the bivalents, but faintly (Text-fig. 5 ~d P1. ] 9, fig. 2). Small heterochromatic segments are found in most chromosomes. The early diplotene Mvalents can be studied much more satisfactorily in aeeto-earmine '~gash Preparations than in sectioned material. The ehiasmata are distributed at random;

482 Studies on the structure of chromosomes

there is no localization. The ' lamp-brush ' fibres are similar in size, shape and st~b,:~ reactions ~o ~hose of the pachytene ttu'eacts, and al)pe'~r to be eommcted with ti~e chrome g. meres (Text-fig. 6). At early diakinesis the 'laml)-brt~sh' fibres are ~],ore distinct and prominent, bu t do not stain much brighter than at the earlier stages ( [e~t fi- P1. 19, figs. 1, 4-). They are observed equally well in fixed and sectioned.material anfia aceto-earmine-treated squash preparabions. That the fibres belo~g to i, he ehroraoso~esi ~ evidenced by the fac~ that diakinetie bivalents can be ~'orced out o[ cells iu s,u~ preparations and are still found to re~ain them (tlmir ' ' "~ ~a laml)-I)rush fi )r(,s) Duri " "' ng lat~ diakinesis (or ]?rometaphase) these fibres cease to be observed, alt]mugh t]~e contour of

Text-fig. 3.

I 1 Text-fig. 4.

/

Text-llg. 5.

Tcxt-lig. 6. Text-fig. 3. Meiotic prophgse nucleus, showing finely beaded lcptoteue threads trod hctcropyenotlc X-chromo.

some. ]3elfin-Allen, Mann's methyl-blue eosin. • Tex~-fig. 4. Zygotene nucleus, showing p~iring ~nd the heteropycnotic X-chromosome. Ace~o-cm'mino squash.

x 2000. Text-fig. 5. Meioi~ic prot)hase nucleus, showing pachytcne chromosomes, eq.ippcd with 'l,.~mp.brush' tlbr~,

~nd heterot)ycnotie X-chromosome. :Bouin, Mann's methyl-bhlc eosin. • 2500. Text-fig. 6. Two diplo~ene chromosome pairs, showing chiasm,~t,~ and 'lamp-hrush' processes. Accto-Qarmhe

squash, x 2000.

the bivalents is still somewhat irregular (Text-fig. 7 b and P1.20, figs. 7, 8). It is diiliou]~ to ascertain whether they are lost or withdrawn. From diplotene to mct,~l)hasc ~hero is a moderate reduction of chiasmata per nucleus (Text-fig. 7). At i)rometal)l,ase th.e shor~es~ bivalents have at least one chiasma, and the bigger ones two chi~smata (171.20, figs. 7, 8).

Throughout the prophase, the X-chromosome is ht ~t highly condensed state, and s~ins deeply. I t is never found to possess ' lamp-brush' iibres.

tfirst metaphase. I~,Tine bivalents and one univalent (X-chromosome) are counfecl ~ the first metaphase, as at pachytene, diplotene and di~d<in(~sis. As is usually the case, fhe meiotic metaphase bivalents are oonsicterably smaller than tim s permaf, ogo~lial ~letaphase

M~. D. L . S ~ V ~ S T ~ V ~ 4=83

~0mosomes, owingt~o their greater degree of spiral condensation. There are usually ~ -;n-s ('l'ext~-fig. ~c and P1.20, figs. 7, 8 and 9) wi~h two chmsmata each. The X-chromo- l~;:~s's like the autosomes and is distinguishable from the la~er only by virtue of i~s being u~pairad. The scparatnon of chromosomes is no~ sy~aehronous for all bivalen~s, ~he sh0r~sr ollrOmOS~ ." ,," (nol, no, eessarily including ~he slior~es~), which are held by one ter$i~a.lized elnasma only, begin ~o separate earlier than the bigger ones (Text-figs. 8, 10).

J

8000^^ |

R@OOAA -

TexM]g. 7. (a) Selmra.t(~, drawings of ghe early dialdnetic bivalengs and X-chromosomes from gwo nuclei. Bouin, Namfs me~llyl-bluo ec).sin, x 2000. (b) Separage drawings oflage dialdnegic bivalengs and X-chromo- somes from I;wo nuclei. Corrosive sublimage-acegio, Unna 's mixgure of pyronin and meghyl green, x 2000. (~) Chromosomes of lirs~ mcil)~ic megaphase nucleus. Separagely drawn. Boule, )'Ialm's meghyl-blue e0Sill, x 2000.

~'irst ana phase. A.t~ anaplmse the chromosomes have their ehromatids widely separated as~hey move t~owards the poles (P1. 19, fig. 3 and Text-fig. 9). The X-chromosome goes to one pole or t~he otSer.

~%cond 'meiolic di'visio~. The interkinetie period is exceedingly short, and the chromo- SOmes ~L~ t~he second ,nci~aphase possess more or loss the same size and appearance as at the ~s~ anaphasc. They still h'~ve their ehromatids separated, which come together when they are arranged on l)}.l.e equator of the spindle (Text-figs. 11, 12 and 13). Even then ~0nae sisger ehromat~ids may remain diverging. In polar view the chromosomes are found arranged ia a circle. One member of the complement is usually enclosed in the ring, often

484 Studies on the structure of eh~'omosomes

Text-fig. 8. Separa~o ch'awings of the firs~ meiotic me~phase b iv~en t s h 'om ~hreo nual~[. I~ouin, l~o~nn's methyl-blue eosfil, x 3500.

Text-fig. 9. Jd'irst meiotic an~phase. A supenmmer~ry (S) is present. Bmfin, ~,{~m's methyl-blue eosin, x 3500.

M. D. L. SI~IVASTAV2k 485

#h its arms widely spread out, almost in a straight line (Text-fig. 11). This is not the X4Romosome, as it is ]?resent in cells with nine chromosomes as well as in those with tO ~hromosomes. This arrangement is characteristic and is nearly always observed, and th8 ~lember enclosed is usually the same. Separation of chromatids is normal (Text-

14) In aceto-carmine squash ])reparations the second anaphase chromosomes clearly g' _1 'their duplex character The highly coiled chromonema, in which a major spiral is

r0ge~ " " s@erimposed on a distinctly observed lninor spiral, lies in a matrix (Text-fig. 15). The 01~0monema and matrix both stain with carmine, but the chromonema is distinguishable ~0m the ground substance, possibly on account of its greater density. In sections, liow- ~v~r, the anaphase chromosomes do not betray their dual character.

At tclopliase also the structure of the chromosomes can sometimes be observed. In pr0perly fixed and stained preparations they show distinctly the two components of

4

Text-fig. 10. Text-fig. 11. Text-fig. 12.

Te~fig. 10. Fh'st meiotic metaphase , showing the normal orientation of the supermunerar ies (S) on the spindle. �9 Thenormal members of the complement are not all ch'awn. ]3ouin, l~Earm's methyl-blue eosin, x 1500.

Tex~-fig. 11. Second meiotic metaph~se. Nine normal members of the ctn'omosome complement form a ring, and one member is centrally placed. The ring contains two supermlmeraries (~). ]3re• l~Eann's methyl-blue eosin, x 2000.

Text, fig. 12. Same as Text-fig. 11 except tha t there is only one supernumerary , x 2000.

which they are composed, namely, a still highly coiled, one-strand chromonema and a comparatively faintly staining matrix in which it is embedded (Text-fig. 16).

On using any single stain generally employed for such studies, like gentian violet, hematoxylin or basic fuchsin, the autosomes stain more or less uniformly, throughout the meiotic cycle, only more densely at metaphase and anaphase than at prophase, which is accounted for by the greater condensation of the chromosomes at metaphase and the higher charge of nucleic acid. By using Mann's methyl-blue eosin, however, an extra- ordinary fac~" is noted. The metaphase, anaphase and the early telophase chromosomes ~e found to be consistently eosinophil and stain bright red, whereas at prophase and late ~10phase they stain ~dth methyl blue only. The X-chromosome is strongly eosinophil throughout meiosis, and is observed as a red mass in the spermatid nucleus, whereas the autOSomes are reduced therein to small amorphous patches, staining faintly ~dth methyl blue. The nuclei of spermatozoa are wholly eosinophil. At late diakinesis the bivalents ee still blue, but may be red in parts. ]garly metaphase bivalents rarely stain purple. There is hardly any doubt that some reversible qualitative change in the chromosome, a~q.: t:D-o . . . . . . e.r ;~-~, ~:vr~o!u~ :q~,2 )roJ)b ~so t:r,. "u-~t-,fl~o~e~ . , . . . . dc,z]i,;~ ,_1.:..,. ~ , , lnbe:731._r2]n n

486 Studies on the structure of chromosomes

Unna's mixture of pyronin and methyl green is held to stain desoxyribm~acleie acid green and libonucleic acid red (vide Pollist.er & Ris, 194=7). It has been found to give inconstant results on this material. The nuclei of spermatozoa, however, always stai~ violet, indicating the presence of both the nucleic acids.

Super In the spermatocytes of two individuals, two small super~mmeratie~ have been observed at the first metaphase (Text-fig. 10, and P1. 20, figs. 9, ]0). In so~ae only one supernumerary has been found. In most individuals, however, the spermat0. cytes have the normal complement. It is not quite easy to determine whether they ate single chromosomes or bivalents, especially as they have not been observed at Sperms.

Text-fig. 14. Text-fig. 13.

Text~fig. 15. Text-fig. 16.

Text-fig. 13. Second meiotic metaphase, showing the arrangement of chromosomes on thc sph~dJe. Bouin, ]~aml's methyl-blue eosin. • 2000.

Text-fig. 14. Second meiotic ~naphase, showing the separation of cha'omosomes on the spindle. Bouin, i~[ama% methyl-blue oosin, x 2000.

Text-fig. 15. Five second meiotic anaphase chromosomes, showing the major and minor spirals in thB chromonem~ta, and the matrix. Acoto-c~rmine squash. Drawn frec-h~md.

TexLfig. 16. Second meiotic telophase mmlous, showing cln.omosomes which are becomhag diffuse. 0hremo- nematic spiral and the matricat substance are observed. Benin, h'[ann's methyl-blue cosin, x 2000.

togonial metaphase and meiotic prophase. From its configuration one looks to be a biva- lent (Text-fig. 10). They remain separate, and show no tendency to come into relation with tlie X-chromosome or other chromosomes. At metaphase they are arranged normally on the equator of the spindle (Text-fig. 10), and have been observed, one or both, atthe second metaphase (Text-figs. 11, 12). There are not enough data available to allow a decision on the point whether they are sporadic or constitute a part of the uorm~l com- plement of a separate strain in the species. The second alternative is quite possibl% especially in view of the fact that the members of this species are visibly of two dis~ia0g types, not only with regard to their external features, but also in respect of the .shape and size of the testes, and the supernumeraries have been found confined to a single strata. Another peculiarity, not observed so often, but also confined to this strain, is the existence

M. D. L . SI~IVASTAVA 487

d clear vesicles roLmd the metaphase bivalents (P1. 20, fig. 9). This is an artifact, but ~0bably a characteristic one.

Pre,,~zdi,i)loid q. Occasionally giant Sl~ermatocytes with a multiple munber of metaphase bi~alen~s are encount,ered (P1.20, figs. 5, 6). These seem to result not from aberrations of @err~a~ogonial mit,osis, but from the fusion of pachy~ene or diakinetic nuclei, although the first alt,ernat,ive cannot be entirely ruled out, as the entire series of processes have not been ~raced. There are, however, no quadrivalents, and in one giant spermatocy~e the ~uraber of chromosomes at first metaphase has been counted to be forty-five bivalents a~d five univMeuts, ehe X-chromosomes. Both these features point to syndiploidy. I)u~ (1950), incidentally, has observed quadrivalents in Ch~'otogonus sp. I t is hardly possible ~hat these abnormM spermatocytes can become ftmctional spermatozoa.

DISCUSSION

Olvromome~'es. One of the evidences brought forth by Ris (194-5) for repudiating the existence of chromomeres is his observation that the diplotene bivalents do not show 0hromomeres. This is not borne out by my observations on Ch~'otogonus, in which chromo- meres are readily observed at diplotene, especially after aceto-carmine staining. I t is true that t,hey are not clearly observable at diakinesis, but this is hardly surprising in ~dew of t,he great,ly t,hickened state of the bivalents at this stage. The evidence from the material invest,igated by me does not justify the rejection of the chromomere concept.

Ohromosome mal,'rix. A number of observers have described a matrical substance in ~,hich t,he chromonemata lie embedded (Heitz, 1935; Makino, 1936; McChmg, 1941).

It.is, however, writ,es with regard to this substance, that 'probably many a matrix in the li~erat,ure is not,hing but the apparent connexion between faintly staining outer loops, running at, an even distance from the darker core of the chromosome where the chromo- nerna~a overlap'. In this way he explains Makino's photographs of cliakinesis and first- metaphase chromosomes of Podisma. This explanation, however, does not hold good in the present, case. Tlle matrix is observed at the anaphase and telophase of the second meiotic division, when the ehromonema is presumably single. Makino & Momma (1950) have shown t,hat, in t,heir material each chromonema consists of two threads. However, the ~hreads are so closely applied to each other that they would act as one. In any case they do not, form t,wo separately coiled chromonematic systems, so as to make t{is's explanat,ion applicable to anaphase and telophase chromosomes. I t may be noted that ~he mat,rical subst,ance is observed, not only in sectioned material, but also in aceto-carmine squash preparat,ions, not pretreated in any special way. From his own observations the ~vri~er is convinced t,hat the matrix is not misinterpreted gyres of chromonemata, but

definit,e sul)st,ance, apart from the latter. 'Lav~'p-br~ish '.fibres. 'Lamp-brt~sh' chromosomes were first described in the yolky eggs

of fishes, amphiMans, Sam'opsida and arthropods, during the growth period. In rome germ cells t,hey have been described in certain grasshoppers by Ris (194-5) and Hsu (194-8) and were el)served 1)y Hearne & Huskins (1935) and many earlier investigators (vide Wilson, 1928).

The diaMuet,ic chromosomes of the spermatocytes of Ch,rotogonus possess ' lamp-brush' fibres which resemble those of Hip~)iscus, as showa by Ris (194-5). These processes, h0wevcr, are found also on pachygene and diplotene threads, as also described by Hsu (1948) in Phlaeoba. i~.fwmata and Catantops humilis. The fact that the hair-like processes

488 Studies on the st~'ueture of chromosomes

in Chrotogonus are always much more faintly stained than the main body of t,he diPlotaae threads, would, on the explanation of Ris, be due to the overlapping of t, he t, wo ch~o~0. nemata on each side of the diplotene loop. This, however, is hardly likely," for ~t di~lt ~ ote~' the disparity between the chromosome body and the hairs, ,with regard ,~o ~he}r stair~ia~ capacity, is great. Also, although these processes mmmare ~ne appearance of toons t~ emmot be traced into actual complete loops under the microscope, especially i~-'ae~: carmine squash preparations, where they appear like processes ending freely. In Hstfs (194-8) figures these processes are shown as big fibres, considerably bigger t;han the gi~h of the main body of the chromosomes. As the ehromonemata undergo increasing spi~al condensation from early paehytene to late diakinesis one would expect; t;he lateral processes to increase appreciably in length on the assumption that they are gyres of the ehromonemata. Nothing like this happens in Uh,rotogonus. (According t;o .[-[stt, althotlgh at diakinesis the ehromatids are considerably shortened and bear elongat;od Processes, there is no spiralization of ehromouemata yet, which occurs at lata~, prometaphase.) Moreover, the lateral fibres on this assumption should not be well formed at; paehytene, as in actual fact happens. Further, in Uh,rotoqonus these processes are fai fly widely spaeecl at diakinesis. Such eonfigural~ions of the diakinetie bivalents are also dopiet;od by other investigators including gis (19t5). This would mean on the explanation of ]~is that the helices of the spiral of chromonemata are loosely t~rranged even at diakilmsis. The lateral processes have been observed by the present writer and also by Hsu (] 948) at the [our- strand diplotene stage, when the ehromatids of each partner in a bi~alent are ~4sibly separated and[ can be followed individually. The hypothesis of Ris ca,mot; explain all these facts.

As for Serra's view that the ' lamp-brush ' processes represent rods of ,mdeoplasm deposited on the gyres of the ehromonemata, this is hardly feasible, as t;h c ,mcleoplasm is clear and almost unstaining during the late meiotic prophase, whereas t;he processes s~ain distinctly. Besides, they seem to be an integral part of the chromosomes and can resist considerable interference.

Hsu's (1948) approach to the understanding of the problem is entirely different. He writes: 'Where the intracellular conditions favour the exhibition by ~;hc X of a l~mp- brush structure and cause the autosomes to be compactly coiled [as in t;he early sperma- togonial divisions] negative heteropyenosis of the X will result. Where ~;he conditions are reversed, as in the meiotic prophase, the X-chromosome remains eoml)aet;ly coiled, while the autosomes all show the typieaI lampbrush struetm'e; under these circumstances the X may be described as positively heteropyenotie. ' This hypothesis, howm~er, has certain weaknesses. The spiral condensation of the ehromatids and ~;he 'lamp-brash' processes exist simultaneously in the diakinetie chromosomes of Chrologonus and of several other grasshoppers. This would not be expected on the basis of .[-[su's hypothesis. Then the negatively heteropyenotie X-chromosome is not shown to possess 'lamp-brash' fibres (White, 19~=0; R ay Chaudhuri & Durra, 19~i7; White & Niekcrson, ]95]). ttsu, however, does not enter into the question whether these processes arc derix~ed from ~he matrix or kalymma, or are derived fl.om the gyres of the ehromonemai;a t;hemsc]ves. The lateral processes, according to him, result from increased synthesis of ,utelcopro~ein, bu~ as both the matrix and the ehromonemata have nueleoprotein, the origin of t;he fibres remains unexplained. In the opinion of the present writer these processes are derived from the matrieal substance (Serra's peripheral nueleoprotein) as Painter suggcsged (] 9~1),

M. D. L. S~rVASTAVA ~89

~?t~ ~he process of 'tamp-brush' formation is identical with endomitosis as seen in the ~rse ceils of cer~a.iu insects (Painter, 19~0) is doubtfill, for these processes cannot be . silifi~bly cousidered as representing full chromosomes: J~ teroch,ro'~at'i~at.ion I t has been recent] shown b several investigators that the go, ' . . . . . . Y ' Y l~et.srochromatic chronmsomes or parts of elu'omosomes are tighbly coiled, when the oachromatic chromosomes remain uncoiled (Shinke, 1937; Coleman, 1940, 19~1, 194-3; 1?llJte, 19'-10 ; Ills, 19d 5 ; Miekey, 19~6 ; Makino & Montana, 1950). In view of the fact that ~hs euchro|natic chromosomes stain the deepest when coiled ~o the highest degree, ~ameIy, at metaphase, this ~s easily understandable and could be reasonably expected ove~ without a direct knowledge. For condensation of the ehromonemata would bring ab0ub a greater concentration of the desoxyribonueleie acid even if its total quantity ~sre no~ to hmrcase. (According to Pasteels & Lison (1951), there is no increase in the total quantity of desoxyribonueleie acid content at metaphase, which is synthesized at tel0phase.) The important point to notice in this commxion is that the heteroelu'omatie c~0mosomes (or parts of chromosomes) behave differently from the euehromatie eln'omo- s0mes during the spiralization and despiralizagion cycle, which must point to physieo- chemical differences in their composition. Caspersson (194-1) attributes a different protein f~amework to the chrolnonemata of the heteroehromatie sections entailing a differential capacity to synthesize nucleic acid. Dar]ington & La Cour (19~0) also attribute hetero- pycnosis to differential synthesis of nucleic acid. Now Prokofyeva-Belgovskaya (19~7) has shown that heterochrolnatization is merely a change of chromosome cycle. This, in all probability, is the case. But that the differences in the absorption curves of euchro- magic mtd hcterochromatic regions as noted by Caspersson (194~0 a, b, 19~1) indicate merely a 0hange of cycle as Prokofyeva-Belgovskaya asserts, is hardly feasible. Since the ea~iromnent is the same for all chromosomes, a differential behaviour in respect of the cyollc changes must necessarily result from a constitutional difference. The fact that the rabes of reproductiou of euehromatin and heteroehromatin may differ (Sehultz, 19r points to such a basic difference.

Another peculiarity of heteroehromatie chromosomes is non-homologous association. 80kradm: (194:1) described such associations in three species of Edessa (Pentatomidae). They are observed ilt the resting spermatogonia, when the heteropyenotic segments ocelot as deeply st, aiued hunps, and during the meiotic prophase. The association between different bivaleuts is brought about by Feulgen-positive interconnecting threads, some of which persist up to the first metaphase. Such associations have been described by other authors as well. ]-[eizer (1950) has recently described interconnecting threads be~geeu spermatogonial chromosomes and tetrads of two species of Oechalia (Penta- ~0midae), aml ]%a.y Chaudhuri & ~Ianna (1950) between the bivalents in some grass- hoppers. This pheuome~tou had been noticed also by several earlier cytologists (vide Nlso~a, 1928). Are these interconnecting ~hreads parts of ~he ehromonemata, or are ~hey derived from the matrix?

h is interestiug to uote that ]~is actually suggested in an earlier paper (19~2) tha~ the i~ereommcting threads might be formed by the flowing together of the semittuid matrix. gahrader (19~1:1) also abtribu~ed the differential behaviour of heterochromosomes to an earlier deposition of matrix round the chromonema, which may be cohesive in earlier Phases. '.[:l~is is in consonance with the findings of Oaspersson & Sehultz (1938) that heteroehromatiu is especially active in synthesizing nucleic acid. Ig is thus exceedingly

Journal of Gcnclics 52 32

490 Studies on the structure of chromosomes

likely that not only the 'lamp-brush' fibres, but also these interconnectiag threads, joining the spermatogonial and meiotic chromosomes, are derived from the substance and are essentially of the same physico-chemical character, raatrical

In Chrotogonus distinct interconnecting thl'eacts are never observed at any stage a- from casual entanglement of 'lamp-brush' fibres, also noticed by Slizynsld (19An, '. van mouse. ~) la the

Supern~tmer(t,r~es. Supernumerary chromosomes have been obseived~ ' " in or~ho~b._~ ~a

species by many investigators (McClung, 1917; Carothers, 1917; Wenrich, 1917. , ~',obe~." ~ son, 1917; Carroll, 1920; Corey, 1933; Mhlouchi, 193~i; Itch, 1934; [i[ehvig, 19~2; Cole~aaa, 194-8; White, 194=9; Rothfels, 1950). They have also been found in many other groups of insects (vide Wilson, 1928). Their origin in most cases is far from certain. Non.disiltaetb ~ is considered one important factor. Recently, Rothfels (1950) has adduced e~ddeace b show that in an individual of Neopodismopsis abdominalis the supermnneraries a~e all distally deficient X-clu'omosomes. They are strongly heteropycnotic. As regards Ghroto. genus the data unfortunately are not sufficient to permit a reasonably safisfaeio~y conclusion about their origin. Are the supernumeraries fragments of chromosomes? This is hardly possible, as they are oriented regularly on the equator of the spindle and survive up to the second metaphase. In the early meiotic prophase there is only one hete~o- pycnotic chromosome, with which the supernumeraries are not found associated at any stage. I t is quite possible they are remnants of chromosomes which have disappeared ~om the complement.

~UMMAI%Y

The structllre and behaviour of the chromosomes of male germ coils of Ohrotogonus incertus have been studied. At the spermatogonial metaphase nine pah's of autosomes and one X-chromosome are observed.

The leptotene threads are single and beaded, bearing chromomeres, which are granules and not twists in the chromonema.

From pachytene to diakinesis the autosomes bear 'lamp-brush' fibres, which at6 considered to be derived from the matrix.

The X-chromosome is found in a condensed state throughout the prophase. The matrix has been clearly distingtfished from the chromonema at the second meio~b

anaphase in aceto-carmine preparations, and at second telophase iu routine preparatbns. Two supernumeraries have been observed, in some nuclei only el,e, at meiotic meta-

phases, and first anaphase. They seem to be confined to a single strain in tile species. Syndiploidy has beeu observed in a few spermatocytes. In one as many as foray-five

bivalents and five univalents (X-chromosomes) have been counted. There are no quadrivalents.

I wish to thank Prof. C. I-I. Waddington, Sc.D., F.R.S., who gave me facilities in the Institute of Animal Genetics, Edinburgh, to complete this work. ] also thank Mr D. Pinkney, who took the photographs.

M. D. L. S~iVAST~VA 491

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EXPLANATION OF PLATES

PLA_'r~ 19 ~igs, 1, 4. [)iakinctic bivalents, showing 'lamp-brush' processes. :Bolfi31, JY[ann's methyl-blue eosin. • 2500. Fig. 2. h[ciotic prophase uueleus, showiug p•chyf, enc t~hreads witch 'l~mp-brush' fibres ~nd het~erochrom~tic

knobs, and the hcteropyeuotic X-chromosome. :Bouin, hf~nn's met~hyl-bhm eosin. • 2500. Fig, 3. ~'irst meiotic anapllase, polar-view, showing nine clu'omosomes. Bmfin, ~Iann's met~hyl-bllm eosia.

x 2500. PLATE 20

Fig. 5, A first meiotic met~aph~sc syndiploid uucleus. Bmdu, ~[~ml's methyl-blue eosin, x 1500. Fig. 6. A first meiotic met~ph~se syndlploid (pent~ploid) nucleus. Bouin, ~[ann's met~hyl-blne eosin, x 1500. ~igs. 7, 8. L~to diakinctfic nuclei, showing ring ~md rod-like bivalent~s ~nd X-chromosomes. The 'lamp-brush'

fibres arc dis~ppcarhlg. Bmfin, h'[auu's met~hyl-blue eosin, x 2500. Fig, 9. Em'ly meiotic met~ph~se nucleus, showing, in ~ddition to t~hc normal members of t~he complements, t~wo

supernumeraries (S). ~Bouin, h'[ann's met~hyl-bluc cosin, x 1500. Fig, 10. ~'ull meiotic met~phase nucleus, slowing t~he normal bivalents, an X-chromosome ~nd t~wo super-

numcr~u'ies (S). x 1500.