Neocentromere-mediated Chromosome Movement in Maize

Neocentromere activity is a classic example of nonkinetochore chromosome movement. In maize, neocentromeres are induced by a gene or genes on Abnormal chromosome 10 (Ab10) which causes heterochromatic knobs to move poleward at meiotic anaphase. Here we describe experiments that test how neocentromere activity affects the function of linked centromere/kinetochores (kinetochores) and whether neocentromeres and kinetochores are mobilized on the spindle by the same mechanism. Using a newly developed system for observing meiotic chromosome congression and segregation in living maize cells, we show that neocentromeres are active from prometaphase through anaphase. During mid-anaphase, normal chromosomes move on the spindle at an average rate of 0.79 μm/min. The presence of Ab10 does not affect the rate of normal chromosome movement but propels neocentromeres poleward at rates as high as 1.4 μm/min. Kinetochore-mediated chromosome movement is only marginally affected by the activity of a linked neocentromere. Combined in situ hybridization/immunocytochemistry is used to demonstrate that unlike kinetochores, neocentromeres associate laterally with microtubules and that neocentromere movement is correlated with knob size. These data suggest that microtubule depolymerization is not required for neocentromere motility. We argue that neocentromeres are mobilized on microtubules by the activity of minus end–directed motor proteins that interact either directly or indirectly with knob DNA sequences. C urrent models suggest that chromosomes move by a combination of forces generated by microtubule disassembly (Inoue and Salmon, 1995; Waters et al., 1996) and the activity of molecular motors (Vernos and Karsenti, 1996; Yen and Schaar, 1996). Microtubule disassembly generates a constant poleward force; while molecular motors can generate force in either poleward or away-from-pole directions, depending on the characteristics of the motor protein. Both plus and minus end–directed microtubule-based motors are localized to kinetochores (Hyman and Mitchison, 1991). Immunolocalization experiments indicate that mammalian kinetochores contain the minus end– directed motor dynein throughout metaphase and anaphase (Pfarr et al., 1990; Steuer et al., 1990). The kinesin-like proteins CENP-E, which has a transient kinetochore localization in animals, and MCAK, which is localized between the kinetochore plates of mammalian chromosomes, are also thought to generate and/or regulate chromosome movement (Yen et al., 1992; Lombillo et al., 1995; Wordeman and Mitchison, 1995).In addition to the molecular motors on kinetochores, several kinesin-like proteins are localized to chromosome arms (Vernos and Karsenti, 1996). Two subfamilies of arm-based motors have been identified in animals: the NOD subfamily (Afshar et al., 1995; Tokai et al., 1996) and the Xklp1/chromokinesin subfamily (Vernos et al., 1995; Wang and Adler, 1995). Both Nod and Xklp1 are required for positioning chromosomes on the metaphase plate, suggesting that they encode plus end–directed motors (Afshar et al., 1995; Vernos et al., 1995). Other evidence suggests that minus end–directed motors interact with chromosome arms. In the plant Haemanthus, a poleward force acts along chromosome arms during metaphase (Khodjakov et al., 1996), and forces propelling chromosome arms poleward have been detected during anaphase in crane fly spermatocytes (Adames and Forer, 1996). Little is known about how poleward arm motility at metaphase–anaphase affects the fidelity or rate of chromosome segregation.The neocentromeres of maize (Rhoades and Vilkomerson, 1942) provide a particularly striking example of poleward chromosome arm motility. In the presence of Abnormal chromosome 10 (Ab10),¹ heterochromatic DNA domains known as knobs are transformed into neocentromeres and mobilized on the spindle (Rhoades and Vilkomerson, 1942; Peacock et al., 1981; Dawe and Cande, 1996). Knobs are primarily composed of a tandem 180-bp repeat (Peacock et al., 1981) which shows homology to a maize B centromere clone (Alfenito and Birchler, 1993). A characteristic feature of neocentromeres is that they arrive at the spindle poles in advance of centromeres; in extreme cases the neocentromere-bearing chromosome arms stretch towards the poles (Rhoades and Vilkomerson, 1942; Rhoades, 1952). A recently identified mutation (smd1) demonstrates that a trans-acting factor(s) encoded on Ab10 is essential for converting the normally quiescent heterochromatic knobs into active neocentromeres (Dawe and Cande, 1996).Here we use neocentromeres as a model for understanding the mechanisms and importance of nonkinetochore chromosome movement. As a part of our analysis, we developed a four-dimensional system for observing chromosome segregation in living meiocytes. Our experiments were designed to determine (a) how poleward arm motility affects the rate and fidelity of chromosome segregation; and (b) whether the mechanism of neocentromere motility is comparable to the mechanism of kinetochore motility.     

Effects of substance P on Na and K transport in perfused main submandibular duct of rat

Substance P (2 and 4 micrograms/kg . min, iv) caused an inhibition of net efflux of Na and net influx of K in perfused main excretory duct of rat submandibular gland. These effects could not be blocked by atropine sulfate. The data suggest that substance P receptors are present in the duct cells and play a role in the regulation of transductal electrolyte transport.     

Replication of chromosomal DNA in cultured abnormal human cells

Summary The replication of chromosomal DNA in a series of abnormal human cell cultures has been studied by means of DNA-fiber autoradiography. In lymphocytes with trisomy 21, in fibroblasts of 45,X; 47,XXX; 49,XXXXY; and 49,XXXXX chromosomal constitution, and in fibroblasts from a patient with xeroderma pigmentosum (De Sanctis-Cacchione syndrome), the rate of DNA replication does not differ from that in normal cells, varying in a single fork from 0.2 to 1.0 m/min with a mean of about 0.6 m/min. In fibroblasts with trisomy 7 the rate of DNA replication is greater, varying from 0.3 to 1.2 m/min with a mean of about 0.8 m/min. The sizes of replication units in all cells examined are from 80 to 500 m with a mean of about 200–300 m.     

Respiratory system of Fusidium coccineum and regulation of biosynthesis of fusidic acid and sporogenesis

Summary The respiratory system, sporulation, and dynamics of alkaline protease formation were studied in three strains of the fungus Fusidium coccineum, differing in their ability to make antibiotics.Oxidative phosphorylation provided most of the energy in high and low activity strains and their respiratory activity was exclusively related to mitochondria functioning.In inactive and low activity strains, the terminal oxidation of reduced equivalents proceeds mainly by the respiratory chain with cytochrome oxidase as the terminal component. In the high activity strain there is a cyanide-resistant alternative pathway which is parallel to the classical cytochrome chain. The complete transition to the use of this pathway coincides with the stage of maximum antibiotic biosynthesis. The induction of the alternative pathway in the high activity strain was not concerned with the inhibition of the cytochrome site of the respiratory chain by fusidic acid. It was shown that the quantity of the antibiotic synthesized and the character of cellular differentiation can be altered by changing the oxidation pathwats used with inhibitors such as chloramphenicol and salicyl hydroxamate.We suggest that there must be common regulation of antibiotic formation, sporulation and induction of the alternative oxidation pathway.     

Functional properties of locust locomotor muscles

The ultrastructure of the muscle fibers and the electrical constants and responses of the membrane to microapplication of L-glutamate and acetylcholine were investigated in the longitudinal flight muscle and the flexor tibiae ofLocusta migratoria migratorioides. The twitch flight muscle differs from the slower leg muscle in the smaller size of its sarcomeres and the lower values of the space attenuation factor of the electrotonic potential, time constant, and resistance of the membrane. Microapplication of sodium L-glutamate at strictly definite points of the fibers of both muscles evoked depolarization responses of the membrane. In experiments on normal and denervated muscle, during microapplication of acetylcholine, changes in the level of the membrane potential were never observed. It is concluded that L-glutamic acid is the excitatory mediator of the twitch and slow muscle systems of insects.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 5, pp. 532–538, September–October, 1977.     

Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: Module evolution of the filamentous phage genome

Summary The nucleotide sequence of the circular single-stranded genome of the filamentous Escherichia coli phage I2-2 has been determined and compared with those of the filamentous E. coli phages Ff(M13, fl, or fd) and IKe. The I2-2 DNA sequence comprises 6744 nucleotides; 139 nucleotides less than that of the N- and I2-plasmid-specific phage IKe, and 337 (336) nucleotides more than that of the F-plasmid-specific phage Ff. Nucleotide sequence comparisons have indicated that I2-2, IKe, and Ff have a similar genetic organization, and that the genomes of I2-2 and IKe are evolutionarily more closely related than those of I2-2 and Ff. The studies have further demonstrated that the I2-2 genome is a composite replicon, composed of only two-thirds of the ancestral genome of IKe. Only a contiguous I2-2 DNA sequence of 4615 nucleotides encompassing not only the coat protein and phage assembly genes, but also the signal required for efficient phage morphogenesis, was found to be significantly homologous to sequences in the genomes of IKe and Ff. No homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the replication genes. Although other explanations cannot be ruled out, our data strongly suggest that the ancestor filamentous phage genome of phages I2-2 and IKe has exchanged its replication module during evolution with that of another replicon, e.g., a plasmid that also replicates via the so-called rolling circle mechanism. Offprint requests to: R.N.H. Konings     

Early stages of formation and dispersal of the temperate flora in the Boreal Region

Budantsev, L. Yu. (Komarov Botanical Institute, Prof. Popov Str. 2, 197376 St. Petersburg, Russia). Early stages of formation and dispersal of the temperate flora in the Boreal Region. Bot. Rev.58(1): 1–48, 1992.—The thesis of this review is that, as stated as early as 1908 by V. L. Komarov, the composition of a flora can be understood only as a process, or separate stage, in the context of migration in time and space of various floristic assemblages and their isolation, as induced by transformation of continental and ocean shapes, changes in climate, and the environment as a whole. Thus the formation of geofloras of the past was influenced by gradually changing environments that determined the spread, patterning, and spatial differentiation of floras and their evolution. Parallel to the more commonly-seen names of eras—Paleozoic, Mesozoic, and Cenozoic—we can speak of the Paleophytic, Mesophytic, and Cenophytic eras. Eras defined in these two ways (by faunistic or by floristic criteria) do not completely coincide. Generally, changes in the flora have, necessarily, preceded changes in the fauna. It is the Cenophytic with which this review is mostly concerned, the era of Angiosperm dominance. The movement of early subtropical and warm temperate floras in the Early Cenophytic, followed by temperate or even boreal floras, as the climate changes, is traced in detail. The regions discussed most fully are the Boreal-Atlantic and Boreal-Pacific, with emphasis on the Angaro-Beringian flora. The disappearance of archaic forms (e.g., cycadophytes) and the gradual predominance of angiosperms is documented. The movements of the floral assemblages in response to environmental changes are mapped and described. The early development and diversification of the boreal temperate flora is considered to have taken place mainly in Angaro-Beringia, associated with the invasive migration of tropical angiosperms from southeastern Asia.     

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.