New zwitterionic butanesulfonic acids that extend the alkaline range of four families of Good buffers: evaluation for use in biological systems.

Four new zwitterionic butanesulfonic acid buffers that are structurally related to four families of Good buffers were evaluated for use in biological systems. These buffers, with pKa values from 7.6 to 10.7, were compared with a variety of other buffers from the same family and with unrelated buffers to determine their effect on enzyme activity and on microbial growth. The activity of four enzymes with optimum pH values in the alkaline range were tested: beta-galactosidase, esterase, phosphodiesterase and alkaline phosphatase. In general, all the Good buffers, including the new butanesulfonic acid buffers, gave good activity; however, there was variation in activity of certain enzymes with certain buffers. Tris, glycine, and phosphate buffers typically showed variation in activity compared to the family of Good buffers. beta-Galactosidase, in particular, showed greater activity with Good buffers than with phosphate or Tris buffers. Similarly, growth of seven bacterial strains was consistent, with a few exceptions, for all the Good family of buffers with Tris often inhibiting growth. Quantitation of alkaline phosphatase conjugated to antibodies is an important tool in many applications in molecular biology. Several Good buffers gave good signals when compared with Tris at pH 9.5 for detection of proteins using alkaline phosphatase-conjugated antibodies.

Developmental control of cell division in leech embryos.

During embryogenesis, cell division must be spatially and temporally regulated with respect to other developmental processes. Leech embryos undergo a series of unequal and asynchronous cleavages to produce individually recognizable cells whose lineages, developmental fates and cell cycle properties have been characterized. Thus, leech embryos provide an opportunity to examine the regulation of cell division at the level of individual well-characterized cells within a community of different types of cells. Isolation of leech homologues of some of the highly conserved regulators of the cell division cycle, and characterization of their patterns of maternal and zygotic expression, indicate that the cell divisions of early leech embryos are regulated by cell type-specific mechanisms. These studies with leech embryos contribute to the emerging appreciation of the diverse mechanisms by which animals regulate cell division during early development.

Regulation of cyclin A mRNA in leech embryonic stem cells.

Leech embryos undergo invariant patterns of cleavage to yield identifiable cells that have characteristic timings of cell division. To elucidate how these cell-specific differences in cell-cycle timing are regulated, we have isolated a leech cyclin A cDNA clone and determined the patterns of cyclin A mRNA localization in identified cells of Helobdella leech embryos. The intensity of cyclin A mRNA hybridization staining is invariant in most cells but fluctuates systematically in the embryonic stem cells. During the repeated cell divisions of the stem cells, the intensity of cyclin A mRNA staining is high during the early part and low during the later part of each cell cycle. The increased staining at the start of the cell cycle is not due to accumulation of newly synthesized cyclin A mRNA, since the pattern of staining is not affected by inhibition of transcription. Treatment with protease prior to hybridization equalizes the intensity of cyclin A mRNA staining throughout the cell cycle, presumably by the removal of blocking proteins. Thus, the levels of maternal cyclin A mRNA remain constant during cell cycles of the embryonic stem cells, but accessibility of cyclin A mRNA to the hybridization probe fluctuates during each cell cycle. We suggest that the events underlying the fluctuating accessibility of cyclin A mRNA to the hybridization probe may be involved in regulating the translational availability of cyclin A mRNA throughout the cell cycle or protecting nascent cyclin A protein from degradation during the destruction period.

Unequal cleavage in leech embryos: zygotic transcription is required for correct spindle orientation in subset of early blastomeres.

Leech embryos undergo invariant sequences of equal and unequal cell divisions to give rise to identifiable progeny cells. While many of the early cleavages are under maternal control, the divisions of a subset of early blastomeres (the large cells of the D' lineage) are perturbed after the inhibition of zygotic transcription. Analysis of the different types of cells produced in embryos injected with the transcriptional inhibitor, alpha-amanitin, revealed that the symmetry of cell division is perturbed in these large D'-derived cells during this early period of development. These cells, which would normally undergo a series of equal and unequal cleavages, always undergo equal cleavages after the inhibition of zygotic transcription. It appears that zygotically transcribed gene product(s) are required in the large cells of the D' lineage to orient the mitotic spindles properly for these unequal cell cleavages.

Expression of the cell cycle control gene, cdc25, is constitutive in the segmental founder cells but is cell-cycle-regulated in the micromeres of leech embryos.

The identifiable cells of leech embryos exhibit characteristic differences in the timing of cell division. To elucidate the mechanisms underlying these cell-specific differences in cell cycle timing, the leech cdc25 gene was isolated because Cdc25 phosphatase regulates the asynchronous cell divisions of postblastoderm Drosophila embryos. Examination of the distribution of cdc25 RNA and the zygotic expression of cdc25 in identified cells of leech embryos revealed lineage-dependent mechanisms of regulation. The early blastomeres, macromeres and teloblasts have steady levels of maternal cdc25 RNA throughout their cell cycles. The levels of cdc25 RNA remain constant throughout the cell cycles of the segmental founder cells, but the majority of these transcripts are zygotically produced. Cdc25 RNA levels fluctuate during the cell cycles of the micromeres. The levels peak during early G2, due to a burst of zygotic transcription, and then decline as the cell cycles progress. These data suggest that cells of different lineages employ different strategies of cell cycle control.

Transcription in leech: mRNA synthesis is required for early cleavages in Helobdella embryos.

Zygotic transcription was analyzed in embryos of the glossiphoniid leech Helobdella triserialis by autoradiographic detection of tritiated uridine incorporated in the presence or absence of low concentrations of alpha-amanitin. RNA synthesis was first detected after the second cleavage and alpha-amanitin-sensitive RNA synthesis was first detected during the divisions yielding the embryonic stem cells, or teloblasts. RNA synthesis increased as development progressed, and the bulk of alpha-amanitin-sensitive RNA synthesis was found in two classes of cells, the blast cells, which are the progeny of the teloblasts, and the micromere-derived cells. The time during which zygotic gene products are required was determined by observing the developmental consequences of alpha-amanitin exposure. Zygotes microinjected with alpha-amanitin underwent the first several cleavages with normal timing and symmetry, but underwent aberrant cleavages and produced supernumerary large blastomeres during the time that the control embryos generated teloblasts. Once the teloblasts were formed, the microinjection of alpha-amanitin did not affect the production of blast cells by the teloblasts, but it did block the divisions and movements of the blast cells and the micromere-derived cells. These data suggest that zygotic transcription is activated during the early cleavages of Helobdella embryos and that newly synthesized transcripts are required for the generation of teloblasts. Thus, there is an early, critical period of messenger RNA synthesis essential for teloblast production that is distinct from the later phase of messenger RNA synthesis required for cell divisions and cell movements during gastrulation.

The durations and compositions of cell cycles in embryos of the leech, Helobdella triserialis.

When tritiated thymidine triphosphate ([(3)H]TTP) or its immunohistochemically detectable analogue, bromodeoxyuridine triphosphate (BrdUTP), is injected into blastomeres of leech embryos it passes throughout the entire embryo and is rapidly incorporated (within 2 min after injection) into nuclei of cells synthesizing DNA (S phase). In the same embryos a DNA-specific stain can be used to identify cells in mitosis (M phase) or nonreplicative interphase (G(1) or G(2) phase) on the basis of nuclear or chromosomal morphology. Using this procedure, we have determined the lengths and compositions of the mitotic cell cycles of identifiable cells in early embryos of the leech, Helobdella triserialis, and have analysed how the cell cycles change during the first seven stages of development. The relatively short cell cycles of the early blastomeres comprise not only phases of M and S, but also postreplicative gap (G(2)) phases. The lengthening of the cell cycles that occurs as development progresses is primarily accomplished by an increase in the length of G(2) and secondarily by an increase in the length of S and,in some instances, the addition of a prereplicative gap(G(1)) phase; M phase remains relatively constant. These data suggest that the durations of the cell cycles of embryonic cells are regulated by a variety of mechanisms.

Early differences between alternate n blast cells in leech embryo.

Segmentally iterated tissues of the mature leech comprise five distinct sets of definitive progeny that arise from chains of blast cells (m, n, o, p, and q bandlets) produced by five bilateral pairs of stem cells (M, N, O/P, O/P, and Q teloblasts). In each n and q bandlet, two blast cells are needed to generate one set of hemisegmental progeny, and two alternating classes of blast cells (nf and ns, qf and qs) can be distinguished after their first divisions. Furthermore, two distinct subsets of definitive N and Q progeny exist within each hemisegment. Here we first show that there is fixed correspondence between the class of blast cell and the subset of final progeny: ns cells contribute mainly anterior ganglionic neurons and epidermal cells; nf cells contribute mainly posterior ganglionic neurons, peripheral neurons and neuropil glia; qs cells contribute both ventral and dorsal progeny; and qf cells contribute only dorsal progeny. Second, ablation studies indicate that the two classes of n blast cells do not behave as an equivalence group in the germinal band. Finally, we show that the cycles giving rise to nf and ns blast cells differ. These data suggest that cellular interactions within the germinal band may not be critical in establishing the distinct nf and ns cell fates and that, conversely, differences between the two classes of n blast cells may be established at birth.

Subcellular distributions of calcium/calmodulin-stimulated and guanine nucleotide-regulated adenylate cyclase activities in the cerebral cortex.

The subcellular distribution of Ca2+/calmodulin-stimulated adenylate cyclase activity was studied in comparison with that of guanine nucleotide-stimulated cyclase activity. The distributions of these activities were similar among the crude fractions but differed among the purified subsynaptosomal fractions. The specific activity of Ca2+/calmodulin-stimulated cyclase was highest in a light synaptic membrane fraction, which has few, if any, postsynaptic densities, whereas that of guanine nucleotide-stimulated cyclase was highest in a heavier synaptic membrane fraction rich in postsynaptic densities. These results suggest that the Ca2+/calmodulin-stimulated cyclase has, at least in part, a different cellular or subcellular location than the guanine nucleotide-stimulated cyclase.

Molybdate permits resolution of untransformed glucocorticoid receptors from the transformed state.

The presence of 10 mM sodium molybdate has a profound effect on the physical behavior of glucocorticoid receptors submitted to ammonium sulfate precipitation or to DEAE-cellulose chromatography. Molybdate inhibits the inactivation of the unoccupied receptor and prevents the transformation of the steroid-bound receptor that occurs when rat liver cytosol is precipitated with ammonium sulfate. The transformed glucocorticoid . receptor complex is precipitated at 30 to 35% ammonium sulfate, whereas the unoccupied receptor and the untransformed steroid-bound receptor are precipitated at 45 to 55% of ammonium sulfate saturation. The untransformed steroid . receptor complex precipitated at 45 to 55% ammonium sulfate saturation in the presence of molybdate can undergo temperature-mediated transformation when it is redissolved in buffer without molybdate. The presence of molybdate in both the loading buffer and eluting gradient during DEAE-cellulose chromatography prevents the transformation of steroid-bound receptor to a less negatively charged, DNA-binding state which otherwise occurs during the chromatographic procedure. In the presence of molybdate, DEAE-chromatography yields a 33-fold purification of the untransformed steroid . receptor complex.