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Fourteen prometaphase kinetochore microtubule bundles have been examined in electron micrographs of serial sections . The majority (54%) of the microtubules extend from the polar region towards the kinetochore but do not end in the kinetochore proper. Rather, they stop short of the kinetochore (21%), graze the kinetochore (19%), or pass through the kinetochore (9%), displaying a free end distal to the pole . Other microtubules that make up the kinetochore bundle include: kinetochore-to-pole microtubules (24%), chromosome-to-pole microtubules (5%), pieces with two free ends (14%), and those microtubules with one end in the kinetochore and a free end distal to the kinetochore (9%) . We conclude that the majority of the microtubules in the kinetochore bundle are most likely of polar origin rather than having been nucleated at the kinetochore.Prometaphase-I kinetochores can display any one of four patterns of microtubule connections with the poles, but the pattern of microtubule connections is not always correlated with kinetochore position . For instance, a kinetochore directly facing one pole may have microtubule connections with both poles while a kinetochore positioned 90°to the spindle axis may have microtubules running towards one pole only .Kinetochore orientation during prometaphase of meiosis can be very complex . The homologous kinetochores could theoretically participate in at least four orientation configurations: (a) unipolar orientation of homologous kinetochores ; (b) bipolar orientation of homologous kinetochores; (c) unipolar orientation of one pair of sister kinetochores and bipolar orientation of the homologous pair ; and (d) bipolar orientation of both pairs of sisters (1 cf. 8) .Bivalents, under normal conditions, usually achieve stable bipolar orientation before anaphase segregation . However, analyses of time-lapse ciné records of live cells in prometaphase demonstrate that brief unipolar orientation of homologous kinetochores is characteristic of some bivalents (1 cf. 18), as is unstable bipolar orientation (3 cf. 18). Mal-orientations are transient and reorientations of the kinetochores lead to stable bipolar orientation.Although prometaphase kinetochore orientations have been frequently described in fixed cells by light microscopy and analyzed in live cells by ciné analyses, little is known about the structural basis for such orientations . That homologous kinetochores are mechanically connected to the same pole in biva-

Prometaphase I chromosome behavior was examined in wild-type Drosophila melanogaster primary spermatocytes. Cine analysis of live cells reveals that bivalents exhibit complex motions that include (1) transient bipolar orientations, (2) simultaneous reorientation of homologous kinetochores, (3) movements not parallel to the spindle axis, and (4) movement along the nuclear membrane. --Kinetochores and kinetochore microtubule have been analyzed for bivalents previously studied in life. The results suggest that most chromosome motions (complex though they may be) can be explained by poleward forces acting on or through kinetochore microtubules that span the distance between the kinetochore and the vicinity of a pole. The results also suggest that the majority of short kinetochore microtubules may be remnants of previous microtubule-mediated associations between a kinetochore and a pole.

Abstract. Single (individual) bivalents in cultured Drosophila melanogaster primary spermatocytes were detached from the spindle with a micromanipulation needle and placed in the cytoplasm. Such bivalents are prevented from rejoining the spindle by a natural membrane barrier that surrounds the spindle, but they quickly orient as if on a spindle of their own and the half-bivalents separate in anaphase. Serial section electron microscopy shows that a mini-spindle forms around the cytoplasmic bivalent, i.e., the microtubule density in the vicinity of the bivalent is much greater than in other cytoplasmic regions. This microtubule population cannot be accounted for solely by kinetochore nucleation and/or capture of microtubules. Furthermore, the mini-spindles frequently form at odd angles to the main spindle, so that at least one pole has no relationship to the poles of the main spindle.We conclude that a bivalent, or factors that become associated with the bivalent as a result of the manipulation, can either stabilize microtubules or promote their assembly. The bivalent activates latent microtubule organizing centers, or alternatively, polar organizing material has been passively transported from the main spindle to the cytoplasm by the micromanipulation procedure. MOST investigations of mitotic spindle morphogenesis in animal cells have emphasized the role of microtubule organizing centers, the centrosome, and the kinetochore. The extent to which centrosomes and kinetochores determine when and where microtubules will be positioned in a cell is only beginning to be understood (7,12,13,15). Although the importance of organizing centers is apparent, it is unlikely that all specificities for microtubule arrays are controlled by them. For example, while the mechanisms involved in microtubule length determination in vivo remain unknown, it is clear that microtubule length is controlled by something beyond tubulin concentration (1). Recent evidence suggests that chromosomes have an active role in spindle morphogenesis in addition to the role of their kinetochores in the origin of kinetochore microtubules. Chromosomes stabilize or promote the assembly of microtubules (7, 10, 17) and remarkably, the presence of chromosomes activates otherwise inactive centrosomes (7) and pericentriolar material (11). An understanding of how centrosomes, kinetochores, and chromosomes interact to organize a functional spindle apparatus will be essential if mechanisms of spindle morphogenesis and chromosome movement are to be resolved.Cultured Drosophila melanogaster primary spermatocytes (6) are well-suited for micromanipulation experiments bearing on spindle formation. In contrast to most higher eucaryotes, several layers of membranes surround the spindle and separate it from the cytoplasm (24). In addition, the membrane layers are circumscribed by a dense layer of mitochondria. Outside of this mitochondria-membrane-spindle complex, the cytoplasm is relatively free of large cytoplasmic organelles. Therefore, when bivalents are detached fro...

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rasshoppcrs \rere given an acute dose of X-rays, injected with Ht b n~i d i n e and thereafter lnaintained at 42°C. Autoradiographic analysis of testicular tubules taken at daily intervals after injection gave meiotic stage durations. T h e mean chiasll~a frequency n.as reduced 4 days after the hoppers \vere placed at +lo, in both X-rayed and non-irradiated aninials, and remained low in the non-irradiated animals throughout the experiment. In the X-rayed animals the chiasma frequency rose to control levels on the 6th day and dropped again thereafter. Analysis of the data indicates a period ending approxi~nately at zygotene during 15-hich chiasma frequc~lcy can be reduced by high temperature. T h e chiasma frequency can be increased by irradiating leptotene-zygotene. T h e end of the heat and X-ray sensitive stages are ternporally d~s t~n c t . Univalents \ \ w e never produced by the hear treatment. Changes in chiasma frequency are restrictetl to the four pairs of larger chromoson~es where multiple chiasniata are com~nonly found. Hence, the presence o r abscncc of chiasniata sho\vs a con~ples interaction \vith the physical environment.Little is known about thc mechanism of gcnctic crossing-over in higher organisms. In fact, thcre is e\.en disagrecmcnt as to thc time during ~nciosis when crossing-over occurs (Grcll and Chandlcy, 1965;Henderson, 1966). Since chiasma formation and gcnetic recombination are usually considered to be manifestations of the same cvcnt (Brov n and %ohar\r, 1955), an understanding of chiasma formation should clucidatc the problcn; of crossing-otrcr.Production of chiasmata is affected b y both y-:111d X-radiation (La~vrcnce, 196 la, 1961 b; i\lathcr, 1934; Westerman, 1967). At lcast thrcc radioscnsitive periods affecting chiasma formation occur during meiosis and one during premeiotic mitoses. 1,awrence (1961) demonstrnted a decrcasc in chiasma frequency whcn ./-radiation was applied during the prc-inciotic D N A synthetic pcriod and an increase when the treatment was given during latc zytotcne or early pachytene in both Liliz~7/1 1 0 1 1 g i f l o~~1~~1 and Tmdescn/lti;l pnl[~dosn. Similar results werc obtained by Westerman ( 1 9 6 7 ) using X-radiation on thc dcscrt locust, Schistoce~ca greynr-in, with the exception that the increase occurrcd when the trcatmcnt nlas given during leptotcnc-zygotenc. ,\/lather (1934) and Wcsterman (1967) also notcd a significant increasc in chi:islna frcqucncy \vhen X-radiation was applied during pre-mciotic mitoses.Heat shock also affccts chiasma production in S. ,yrega~in (tlenderson, 1962, 1963, 1966) and in a grasshopper, Go?linen nzlstrnlinsiae (Peacock, 1968).A drastic decreasc in chiasma frequency occurs when cither S. gre,qar-in or G. azcstrnliasiae is subjected to a temperat;rc of 4 0°C . 'The heat-sensitive period appears to be latc ygotenc-early pachvtenc.

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