Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes using dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex, or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length–dependent pulling forces, which move and orient centrosomes.

Scrape loading and sonication loading are two recently described methods of introducing macromolecules into living cells. We have tested the efficacy of these methods for transfection ofmammalian cells with exogenous DNA, using selection systems based either on resistance to the drug G418

Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using a-and f-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globinrelated DNA sequences. We detect specific, high-stringency hybridization of the a-globin probe to genomic DNAs of both white-and red-blooded species, whereas the 8-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of a-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes p-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult ,3-globin locus prior to the diversification of the clade.In 1954 Ruud (1) published the first systematic analysis of the "white" blood of an antarctic icefish, Chaenocephalus aceratus. He reported that fresh blood was nearly transparent, contained leukocytes at 1% by volume, but lacked erythrocytes and the respiratory transport pigment hemoglobin. Furthermore, the oxygen-carrying capacity of C. aceratus blood was approximately 10% that of two red-blooded notothenioids. Subsequent investigations extended these observations to other icefish species and revealed that icefish blood contains small numbers of "erythrocyte-like" cells that, nevertheless, are devoid of hemoglobin (2, 3). Thus, limited to oxygen physically dissolved in their blood, the icefishes have evolved compensatory physiological and circulatory adaptations-e.g., modest suppression of metabolic rates, enhanced gas exchange by large, well-perfused gills and cutaneous respiration, and large increases in cardiac output and blood volume-that ensure adequate oxygenation of their tissues (4, 5).We have initiated studies to determine the status of globin genes in channichthyid genomes and to evaluate potential evolutionary mechanisms leading to the hemoglobinless phenotype. Icefishes evolved from the red-blooded Notothenioidei (6, 7), which, in contrast to temperate fishes, are characterized by a paucity of hemoglobin forms (7-10). Adults of the family Nototheniidae (antarctic rockcods) generally possess a major hemoglobin, Hb 1 (-95% of the total), and a second, minor hemoglobin, Hb 2, that differ in their a chains (al and a2, respectively) (11-13). The more phyletically derived harpagiferids and bathydraconids have a single hemoglobin. The trend toward reduced hemoglobin multiplicity in t...

Antarctic icefishes are the only vertebrates that do not have hemoglobin and erythrocytes in their blood. These startling phenotypes are associated in several icefish species with deletions of juvenile and adult globin loci, which in red-blooded teleosts are typically composed of tightly linked pairs of alpha- and beta-globin genes. It is unknown if the loss of hemoglobin expression in icefishes was the direct result of such deletions or if other mutational events compromised globin chain synthesis prior to globin gene loss. In this study, we show that 15 of the 16 icefish species have lost the adult beta-globin gene but retain a truncated alpha-globin pseudogene. Surprisingly, a phylogenetically derived icefish species, Neopagetopsis ionah, possesses a complete, but nonfunctional, adult alphabeta-globin complex. This cluster contains 2 distinct beta-globin pseudogenes whose phylogenetic origins span the entire Antarctic notothenioid radiation, consistent with an origin via introgression. Maximum likelihood ancestral state reconstruction supports a scenario of icefish globin gene evolution that involves a single loss of the transcriptionally active adult alphabeta-globin cluster prior to the diversification of the extant species in the clade. Through lineage sorting of ancestral polymorphism, 2 types of alleles became fixed in the clade: 1) the alpha-globin pseudogene of the majority of species and 2) the inactive alphabeta-globin complex of N. ionah. We conclude that the globin pseudogene complex of N. ionah is a "genomic fossil" that reveals key intermediate steps on the pathway to loss of hemoglobin expression by all icefish species.

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