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C F Lehner

Publications and source records attributed to C F Lehner.

18 recordsLinked to original sources
PubMed1992

Cyclins and cdc2 kinases in Drosophila: genetic analyses in a higher eukaryote.

Cyclin proteins and the kinases with which they associate are encoded by gene families in multicellular eukaryotes. A variety of cyclin/kinase complexes with different functions may exist. We have started a genetic dissection of this complexity in Drosophila. We have done experiments to investigate a potential functional overlap between two kinases (Dmcdc2 and Dmcdc2c) and two cyclins (cyclin A and cyclin B). No functional overlap was observed between the Dmcdc2 and the Dmcdc2c kinases. The phenotype resulting from mutations in Dmcdc2 was not affected by altering the level of Dmcdc2c. Our results concerning cyclin A and cyclin B strongly suggest that these two cyclins have largely overlapping functions. Cell proliferation was observed in the absence of either cyclin A or cyclin B, but not if both cyclins were absent. Cyclin A also has essential functions that cannot be taken over by cyclin B, but these functions appear to be required at defined developmental stages in specific tissues only.

Animals↗
PubMed1992

The Drosophila cdc25 homolog twine is required for meiosis.

We have identified a second cdc25 homolog in Drosophila. In contrast to string (the first homolog identified in Drosophila) this second homolog, twine, does not function in the mitotic cell cycle, but is specialized for meiosis. Expression of twine was observed exclusively in male and female gonads. twine transcripts are present in germ cells during meiosis, and appear only late during gametogenesis, well after the end of the mitotic germ cell divisions. The sterile Drosophila mutant, mat(2)synHB5, which had previously been isolated and mapped to the same genomic region as twine (35F), was found to carry a missense mutation in the twine gene. This missense mutation in twine abolished its ability to complement a mutation in Schizosaccharomyces pombe cdc25. Phenotypic analysis of mat(2)synHB5 mutant flies revealed a complete block of meiosis in males and severe meiotic defects in females.

Amino Acid Sequence↗
PubMed1992

The pebble gene is required for cytokinesis in Drosophila.

Cytokinesis is developmentally controlled during Drosophila embryogenesis. It is omitted during the initial nuclear division cycles. The nuclei of the resulting syncytium are then cellularized at a defined stage, and cytokinesis starts in somatic cells with mitosis 14. However, cytokinesis never occurs in somatic cells of embryos homozygous or transheterozygous for mutations in the pebble gene. Interestingly, the process of cellularization, which involves steps mechanistically similar to cytokinesis, is not affected. Moreover, all the nuclear aspects of mitosis (nuclear envelope breakdown, chromosome condensation, spindle assembly and function) proceed normally in pebble mutant embryos, indicating that pebble is specifically required for the coordination of mitotic spindle and contractile ring functions. The pebble phenotype is also observed, but only with very low penetrance, during the early divisions of the germ line progenitors (the pole cells). alpha-Amanitin injection experiments indicate that these early pole cell divisions, the first cell divisions during embryogenesis, do not require zygotic gene expression. These divisions might therefore rely on maternally contributed pebble function. The maternal contribution from heterozygous mothers might be insufficient in rare cases for all the pole cell divisions.

Actins↗
PubMed1991

Pulling the string: cell cycle regulation during Drosophila development.

The extensive cell proliferation which accompanies the development of multicellular organisms is co-ordinated with other developmental events. Cell cycle progression during embryogenesis is therefore controlled according to developmental stage and developmental fate. In Drosophila, entry into S phase is a constitutive, unregulated event until late in development. Entry into mitosis, however, is the first control point used for differential regulation, and the transcriptionally controlled expression of string (the Drosophila homologue of Schizosaccharomyces pombe cdc25+) directs the precise patterns of the embryonic cell divisions after the onset of morphogenesis. In contrast to string, cyclin proteins are produced in excess and their rate of accumulation does not regulate the time of mitosis.

Animals↗
PubMed1990

The roles of Drosophila cyclins A and B in mitotic control.

We have cloned, sequenced, and characterized the expression of a Drosophila cyclin B gene. The independent evolutionary conservation of A- and B-type cyclins implies that they have distinct roles. Indeed, in mutant embryos deficient in cyclin A, cells that accumulate only cyclin B do not enter mitosis. Thus, in vivo, cyclin B is not sufficient for mitosis. Furthermore, we find that the two cyclins are coexpressed in all proliferating cells throughout development. Though lacking a formal demonstration that cyclin B is essential as it is in other organisms, we propose that each of these proteins fulfills a distinct and essential role in the cell cycle.

Amino Acid Sequence↗
PubMed1990

Drosophila cdc2 homologs: a functional homolog is coexpressed with a cognate variant.

Using probes obtained by PCR amplification, we have cloned Drosophila cDNAs encoding structural homologs of the p34cdc2 cell cycle kinase. Southern blot experiments and in situ hybridization to polytene chromosomes demonstrated that the isolated cDNAs, were derived from two distinct genes, Dm cdc2 (31E) and Dm cdc2c (92F). Northern blot and in situ hybridization experiments revealed that these two genes are coexpressed during embryogenesis and that expression is correlated with cell proliferation. However, despite the similarity in structure and expression, the two gene products differed in functional assays in yeasts. Expression of Dm cdc2 in Schizosaccharomyces pombe and Saccharomyces cerevisiae rescued cell cycle arrest caused by mutations in cdc2+ and CDC28, the genes encoding the p34cdc2 kinase homologs of these yeasts. In contrast, the Dm cdc2c gene product did not restore cell cycle progression. Thus, in addition to the identification of a functional homolog in Drosophila, our results indicate the presence of a closely related cognate of the p34cdc2 cell cycle kinase.

Amino Acid Sequence↗
PubMed1989

Directing cell division during development.

Several evolutionarily conserved proteins constitute a universal mitotic trigger that is precisely controlled during the orderly cell divisions of embryogenesis. As development progresses, the mechanisms controlling this trigger change. Early divisions are executed by maternally synthesized gene products, and in Xenopus they are timed by the accumulation and periodic degradation of cyclin, a trigger component. Later, the zygotic genome assumes control, and in Drosophila, zygotic transcription is required for production of another trigger protein, the product of string. After this transition to zygotic control, pulses of string transcription define the timing of highly patterned embryonic cell divisions and cyclin accumulation is not rate limiting.

Animals↗
PubMed1989

Cloning and sequencing of cDNA clones encoding chicken lamins A and B1 and comparison of the primary structures of vertebrate A- and B-type lamins.

Nuclear lamins are intermediate-filament-type proteins forming a fibrillar meshwork underlying the inner nuclear membrane. The existence of multiple isoforms of lamin proteins in vertebrates is believed to reflect functional specializations during cell division and differentiation. Although biochemical criteria may be used to classify many lamin isoforms into A- and B-type subfamilies, the structural features distinguishing the members of these subfamilies remain to be characterized fully. Here, we report the complete primary structures of chicken lamins A and B1, as they are deduced from cloned cDNAs; in the accompanying paper we present the complete sequence of lamin B2, a second avian B-type lamin. Comparisons of the chicken lamin sequences with each other and with those of other lamins allow us to establish structural features that are common to members of both subfamilies. Conversely, multiple sequence alignments make it possible to identify a number of structural motifs that clearly differentiate B-type lamins from A-type lamins. With this information at hand, we attempt to correlate different biochemical properties of A- and B-type lamins with the presence or absence of specific sequence motifs.

Amino Acid Sequence↗
PubMed1989

A second higher vertebrate B-type lamin. cDNA sequence determination and in vitro processing of chicken lamin B2.

The chicken nuclear lamina is composed of at least three proteins called lamins A, B1 and B2. In addition, putative precursors are transiently expressed during in vivo synthesis of lamins A and B2. Here we report the complete sequence of lamin B2 as it is deduced from a cloned cDNA. Comparison of lamin B2 with lamins A and B1 in the accompanying paper provides definitive proof for the existence of two structurally distinct chicken B-type lamins. Furthermore, we show that in vitro translation of transcripts derived from lamin A and lamin B2 cDNAs yielded polypeptides that were indistinguishable, by two-dimensional gel electrophoresis, from the putative in vivo precursors of lamins A and B2 respectively. However, whereas the lamin A precursor was stable, the translation product of the lamin B2 transcript was processed in the reticulocyte lysate to a polypeptide comigrating on two-dimensional gels with authentic mature lamin B2. This processing event could be inhibited by chelators of divalent cations, i.e. o-phenanthroline and EDTA. Our results indicate that the transiently expressed variant of lamin B2 represent a bonafide precursor, and that two distinct activities are involved in processing of newly synthesized lamins A and B2. Lamin precursors processing is discussed in relation to characteristic differences in the interactions of A and B-type lamins with the nuclear membrane.

Amino Acid Sequence↗
PubMed1989

Expression and function of Drosophila cyclin A during embryonic cell cycle progression.

Cyclin proteins are thought to trigger entry into mitosis. During mitosis they are rapidly degraded. Therefore, mitosis and consequently cyclin degradation might be triggered at a time when cyclins have reaccumulated to a critical level. We cloned and sequenced a Drosophila cyclin A homolog and identified mutations in the corresponding gene. Immunofluorescent staining revealed that cyclin A accumulates in the interphase cytoplasm of cellularized embryos, but relocates to the nuclear region early in prophase and is completely degraded within metaphase. Cyclin A was expressed in dividing cells throughout development, and a functional cyclin A gene was required for continued division after exhaustion of maternally contributed cyclin A. Importantly, the timing of post cellularization divisions was not governed by the rate of accumulation or level of cyclin A.

Alleles↗
PubMed1989

Major nucleolar proteins shuttle between nucleus and cytoplasm.

Nucleolin is a 92 kd nucleolar protein implicated in regulating polymerase I transcription and binding of preribosomal RNA. Another abundant nucleolar protein of 38 kd (B23/No38) is thought to be involved in intranuclear packaging of preribosomal particles. Although both proteins have previously been detected only in nuclei, we conclude that they shuttle constantly between nucleus and cytoplasm. This conclusion is based on monitoring the equilibration of these proteins between nuclei present in interspecies heterokaryons, and on observing the antigen-mediated nuclear accumulation of cytoplasmically injected antibodies. Our unexpected results suggest a role for these major nucleolar proteins in the nucleocytoplasmic transport of ribosomal components. Moreover, they suggest that transient exposure of shuttling proteins to the cytoplasm may provide a mechanism for cytoplasmic regulation of nuclear activities.

Amino Acid Sequence↗
PubMed1988

The fates of chicken nuclear lamin proteins during mitosis: evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum.

In chicken, three structurally distinct nuclear lamin proteins have been described. According to their migration on two-dimensional gels, these proteins have been designated as lamins A, B1, and B2. To investigate the functional relationship between chicken lamins and their mammalian counterparts, we have examined here the state of individual chicken lamin proteins during mitosis. Current models proposing functional specializations of mammalian lamin subtypes are in fact largely based on the observation that during mitosis mammalian lamin B remains associated with membrane vesicles, whereas lamins A and C become freely soluble. Cell fractionation experiments combined with immunoblotting show that during mitosis both chicken lamins B1 and B2 remain associated with membranes, whereas lamin A exists in a soluble form. In situ immunoelectron microscopy carried out on mitotic cells also reveals membrane association of lamin B2, whereas the distribution of lamin A is random. From these results we conclude that both chicken lamins B1 and B2 may functionally resemble mammalian lamin B. Interestingly, immunolabeling of mitotic cells revealed an association of lamin B2 with extended membrane cisternae that resembled elements of the endoplasmic reticulum. Quantitatively, we found that all large endoplasmic reticulum-like membranes present in metaphase cells were decorated with lamin B2-specific antibodies. Given that labeling of these mitotic membranes was lower than labeling of interphase nuclear envelopes, it appears likely that during mitotic disassembly and reassembly of the nuclear envelope lamin B2 may reversibly distribute between the inner nuclear membrane and the endoplasmic reticulum.

Animals↗
PubMed1987

Differential expression of nuclear lamin proteins during chicken development.

By immunocytochemistry, quantitative immunoblotting, and two-dimensional gel electrophoresis, we have analyzed the distribution of nuclear lamin proteins during chicken embryonic development. Whereas no qualitative differences in the patterns of expression of lamins A, B1, and B2 were observed during gametogenesis in either the female or the male germ line, profound changes in the composition of the nuclear lamina occurred during the development of somatic tissues. Most unexpectedly, early chicken embryos were found to contain little if any lamin A, although they contained substantial amounts of lamins B1 and B2. During embryonic development, lamin A became increasingly prominent, whereas the amounts of lamin B1 decreased in many tissues. Interestingly, the extent and the developmental timing of these changes displayed pronounced tissue-specific variations. Lamin B2 was expressed in fairly constant amounts in all cell types investigated (except for pachytene-stage germ cells). These results have implications for the purported functional specializations of individual lamin proteins. In addition, they suggest that alterations in the composition of the nuclear lamina may be important for the establishment of cell- or tissue-specific differences in nuclear architecture.

Animals↗
PubMed1986

The nuclear lamin protein family in higher vertebrates. Identification of quantitatively minor lamin proteins by monoclonal antibodies.

The nuclear lamina, a structure closely apposed to the inner nuclear membrane, is believed to provide a framework important for nuclear envelope integrity and interphase chromatin organization. So far, in mammalian and avian species three major constituents of the lamina, lamins A, B, and C, have been identified. These proteins migrate to characteristic positions on two-dimensional gels, lamin B being more acidic than lamins A and C. Here, we show that the composition of the nuclear lamina in avian and mammalian cells is more complex than previously assumed. When analyzed on two-dimensional gels, the major 66-kDa chicken "lamin B" protein can readily be identified. However, an additional 68-kDa protein migrates to a similarly acidic position. Based on the following evidence, both proteins can be considered as two distinct members of the lamin protein family. First, peptide mapping experiments and immunological criteria demonstrate that these two proteins are not related to each other or to lamin A via postsynthetic modifications or precursor-product relationships. Second, as determined by immunocytochemical techniques, both proteins are located exclusively at the nuclear periphery. Third, both proteins display the biochemical properties characteristic of lamin proteins, i.e. they are resistant to extraction of nuclei with nonionic detergents, nucleases, and high salt. Fourth, both proteins are immunologically related to previously characterized lamin proteins: the major 66-kDa chicken "lamin B" protein shares at least two epitopes with lamin A. However, contrary to what current nomenclature might suggest, this 66-kDa chicken "lamin B" protein is not related to rat liver lamin B, but to a minor component of rat liver pore-complex lamina preparations that had not previously been recognized as a lamin protein. Conversely, the minor 68-kDa component of chicken lamina preparations that had not previously been considered to be a lamin protein is immunologically related to rat liver lamin B. Thus, in addition to demonstrating the existence of quantitatively minor lamin proteins in higher vertebrates, our results caution against assigning structural homologies between lamin proteins from different species on the basis of gel electrophoresis analyses.

Animals↗
PubMed1986

A monoclonal antibody specific for snRNPs U1 and U2.

A monoclonal antibody (D-5) is described which selectively precipitates snRNPs U1 and U2. The antibody was derived from a mouse immunized with extracts from chick embryonic nuclei. By immunoblotting on either total proteins from purified snRNPs U1-U6, U2-U6 or U1 only, we could demonstrate that the monoclonal antibody cross-reacts with the U1 RNP specific polypeptide A and the U2 RNP specific polypeptide B", thereby establishing that the two snRNP proteins share at least one epitope. D-5 precipitates snRNPs U1 and U2 from a variety of species, including man, chicken, mouse, rat kangaroo and Xenopus laevis. It will thus be a useful tool for studying structure function relationships of the two snRNP species in different cell systems.

Animals↗
PubMed1986

Nuclear substructure antigens. Monoclonal antibodies against components of nuclear matrix preparations.

We describe two monoclonal antibodies, I-2 and I-14, which recognize, respectively, proteins of 36 and 40 kD. By immunofluorescence microscopy on chick embryo fibroblasts, both antigens were found to be located within a nuclear substructure which excludes nucleoli and part of the nucleoplasm; hence we refer to these antigens as nuclear substructure antigens. By immuno-electron microscopy on chick liver sections, the I-14 antigen was identified predominantly in clusters of interchromatin granules and in perichromatin fibrils. The two substructure antigens share a remarkable resistance to sequential extraction of nuclei with DNase I, RNase A, non-ionic detergent and high salt, indicating that they constitute part of an operationally defined residual nuclear matrix. Finally, both substructure antigens are virtually absent from the nuclei of adult erythrocytes. These properties suggest that substructure antigens may be involved in RNA transcription, processing or transport, possibly by contributing nucleoskeletal support.

Animals↗
PubMed1986

Biogenesis of the nuclear lamina: in vivo synthesis and processing of nuclear protein precursors.

Utilizing antibodies against lamins A, B1, and B2, we have studied the biogenesis of the nuclear lamina in chicken embryo fibroblasts. (Lamins B1 and B2 have been identified recently as structurally distinct "lamin B" proteins.) We demonstrate that, unique among the nuclear proteins studied to date, lamin A is synthesized as a higher molecular mass precursor. A short-lived higher molecular mass variant (t 1/2 approximately equal to 3 min) accompanying the mature-size protein was also detected in the case of lamin B2 biosynthesis, but no precursor was found for lamin B1. By combining pulse-chase experiments with subcellular fractionation, we provide evidence that synthesis of lamin proteins occurs on free polysomes; subsequently, the newly synthesized proteins become rapidly associated with a crude nuclear fraction. The lamin A precursor is processed within the nucleus with a half-time of about 30 min. Concomitantly, lamin proteins acquire a characteristic resistance to detergent extraction, suggesting their insertion into a submembraneous protein network. The described biogenetic pathway involving precursor synthesis and processing is very unusual for nuclear proteins; it may have interesting implications for the mechanisms of transport and assembly of poorly soluble nuclear proteins.

Animals↗