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R A Laskey

Publications and source records attributed to R A Laskey.

At least 73 records · Page 4Linked to original sources
PubMed1988

Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores.

When injected into the cytoplasm of Vero cells, nucleoplasmin rapidly concentrates in a narrow layer around the nuclear envelope and then accumulates within the nucleus. Transport into the nucleus can be reversibly arrested at the perinuclear stage by metabolic inhibitors or by chilling. Nucleoplasmin-coated colloidal gold particles concentrate around the nuclear envelope of Vero cells or Xenopus oocytes, and by electron microscopy of oocytes appear to be associated with fibrils attached to nuclear pore complexes. Perinuclear accumulation is not observed for the nonmigrating nucleoplasmin core fragment or nonnuclear proteins. We propose two steps in nuclear migration of proteins: rapid binding around the nuclear envelope, possibly to pore-associated fibrils, followed by slower, energy-dependent translocation through nuclear pores.

Adenosine Triphosphate↗
PubMed1988

Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs.

We have studied the pathway of nuclear assembly from demembranated sperm chromatin by fractionating a cell-free system from Xenopus eggs (Lohka, M. J., and Y. Masui. 1983. Science (Wash. DC). 220:719-721). Both the soluble fraction and a washed vesicular fraction are required for formation of normal nuclei that initiate replication in vitro. The soluble fraction alone decondenses chromatin and the vesicular fraction alone surrounds chromatin with membranes. Both fractions are required for formation of nuclear pore complexes. Recombining these two fractions recovers approximately 100% of the nuclear assembly and DNA replication activities. Restricting the proportion of the vesicular fraction slows acquisition of the nuclear membrane and allows observation of immature nuclear pores ("prepores"). These form as arrays around and within the chromatin mass before membranes form. Subsequently membrane vesicles bind to these prepores, linking them by a single membrane throughout the chromatin mass. At the periphery this single membrane is surrounded by an outer membrane. In mature nuclei all membranes are at the periphery, the two membranes are linked by pores, and no prepores are seen. Nuclear assembly and replication are inhibited by preincubating the chromatin with the vesicular fraction. However nuclear assembly is accelerated by preincubating the condensed chromatin with the soluble fraction. This also decreases the lag before DNA replication. Initiation of DNA replication is only observed after normal nuclei have fully reassembled, increasing the evidence that replication depends on nuclear structure. The pathway of nuclear assembly and its relationship to DNA replication are discussed.

Animals↗
PubMed1987

Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts.

The composition and function of histone storage complexes of Xenopus eggs have been investigated using monoclonal antibodies. We show that core histones are contained in two distinct complexes: H2A and H2B are associated with nucleoplasmin, and H3 and H4 are associated with nuclear protein N1. Immunodepletion analyses demonstrate that both complexes are required for nucleosome core assembly by extracts in vitro, the product being a simple sum of the histones from each complex. In addition, the majority of the stored H2A is shown to be an unusual form that migrates close to the position of H3 by SDS-polyacrylamide gel electrophoresis and resembles a variant synthesized in a cell-cycle-independent manner in mammalian cells.

Animals↗
PubMed1987

Chromosome replication in cell-free systems from Xenopus eggs.

Cell-free systems from eggs of the frog Xenopus laevis are able to perform most of the acts of eukaryotic chromosome replication in vitro. This now includes the crucial regulatory step of initiation, which had only been achieved for viral systems previously. Purified DNA or nuclei are able to initiate and complete semi-conservation replication in egg extracts in vitro (Blow & Laskey, Cell 47, 557-587 (1986). Replication does not require specialized DNA sequences either in vitro or in microinjected eggs, but in both systems large templates replicate more efficiently than small templates. In some cases replication can re-initiate, excluding the possibility that replication is primed by preexisting primers in the template preparations. When nuclei are replicated in vitro, only one round of replication is observed in a single incubation resembling the single round of replication observed for purified DNA after micro-injection. The mechanism that prevents re-initiation of replication within a single cell cycle is discussed and certain models are eliminated. Nucleosome assembly from histones and DNA has also been studied in cell-free systems from Xenopus eggs. Fractionation has led to the identification of two acidic proteins called nucleoplasmin and N1, which bind histones and transfer them to DNA. The sequences of both proteins have been determined by cDNA cloning and sequencing. Both proteins are found as complexes with histones in eggs.

Animals↗
PubMed1987

Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals.

Nucleoplasmin is the most abundant protein in the Xenopus oocyte nucleus. It is involved in histone storage and chromatin assembly and it has been used extensively to study the transport of proteins into the cell nucleus. We have isolated lambda gt11 phage containing nucleoplasmin cDNA and have determined the sequence of the entire protein coding region of 200 amino acids for one of the two genes. The translation product of the sp6 transcript of this cDNA has the same electrophoretic mobility as nucleoplasmin and is able to form pentamers. The protein sequence shows remarkable clusters of charged residues including a long polyglutamic acid tract which presumably constitutes the histone binding site. The short C-terminal domain which specifies nuclear entry contains four regions which are homologous to putative nuclear localization signals including two regions of homology to the nuclear migration signal of SV40 large T antigen.

Amino Acid Sequence↗
PubMed1986

Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.

We demonstrate that cell-free extracts prepared from activated eggs of X. laevis by a method similar to that of Lohka and Masui initiate and complete semiconservative DNA replication of sperm nuclei and plasmid DNA. The efficiency of replication is comparable to that in the intact egg. Under optimal conditions 70%-100% of nuclei, and up to 38% of naked DNA molecules replicate completely. Genuine initiation of replication occurs rather than elongation of preformed primers or priming of irreversibly denatured templates. Rereplication of templates is observed under certain conditions. In addition to replicating DNA, these extracts also assemble nucleus-like structures from naked DNA.

Animals↗
PubMed1985

Chromosome replication in early Xenopus embryos.

Early embryos of X. laevis achieve exceptional rates of DNA replication and chromatin assembly. These processes have been studied by microinjecting DNA templates into eggs or by incubating DNA in egg extracts. The egg is able to regulate replication of injected DNA without requiring specialized DNA sequences. Assembly of DNA into the nucleosome subunits of chromatin involves interaction of DNA with complexes containing two classes of acidic protein, namely nucleoplasmin and N1/N2.

Animals↗
PubMed1985

Chromosome replication in early development of Xenopus laevis.

Eggs of Xenopus laevis contain exceptionally large amounts of materials involved in chromosome replication. This maternal stockpile allows an embryo to produce about 80 000 cells in less than 24 h. The adaptations which achieve this involve the mechanisms of both DNA replication and chromatin assembly.

Animals↗
PubMed1983

Phosphorylation of nuclear proteins.

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.

Animals↗
PubMed1983

Induction of chromosome replication during maturation of amphibian oocytes.

After fertilization amphibian embryos replicate their chromosomes faster than bacteria replicate their much smaller chromosomes. During oogenesis, materials are accumulated to sustain these rapid cycles of chromosome replication. Thus growth is uncoupled from nuclear division. Most of the machinery for DNA replication and chromatin assembly is present in the oocyte, which lacks only the ability to initiate on a DNA duplex. After maturation and activation a cell cycle clock is triggered which specifies initiation of DNA replication on endogenous chromosomes, injected nuclei or injected plasmid DNA. The ability to reinitiate replication of a replicated molecule is tightly coupled to the cell cycle clock. Each egg can replicate an amount of DNA equivalent to 500 diploid nuclei in only five hours. However, each egg can assemble an amount of purified DNA equivalent to 12 000 diploid nuclei into regularly spaced nucleosomes in only one hour. The molecular basis of these extraordinary rates of DNA replication and chromatin assembly is considered.

Animals↗
PubMed1983

Tumor promoter TPA increases initiation of replication on DNA injected into xenopus eggs.

The effect of the tumor promoter TPA on the control of DNA replication was assayed by following the regulated replication of DNA microinjected into eggs of the frog Xenopus laevis. TPA increases the amount of replication of injected DNA. Both initiation of replication on parental DNA molecules and reinitiation on previously replicated molecules are stimulated. Interaction with the external membrane appears necessary since injections of high concentrations of TPA into the egg are ineffective, whereas nM concentrations are active in the external medium. Related molecules that lack tumor promoting activity do not affect DNA replication. The effect of TPA on DNA replication was detectable only after the first cell cycle, and TPA cannot induce replication in oocytes, the quiescent stage which precedes the egg. When protein synthesis is inhibited TPA still increases initiation of replication, but does not allow detectable reinitiation cycles. The results suggest that interaction of TPA with the cell membrane is sufficient to increase the efficiency of replication initiation by a mechanism that does not require illegitimate reinitiation within a single cell cycle.

Animals↗
PubMed1982

A polypeptide domain that specifies migration of nucleoplasmin into the nucleus.

Nucleoplasmin is the most abundant protein of the nucleus of Xenopus laevis oocytes. It rapidly enters the nucleus after being injected into oocyte cytoplasm. Partial proteolysis of the nucleoplasmin pentamer reveals two structural domains within each subunit: a relatively exposed "tail" and a protected "core." When all the "tails" have been removed from the pentamer the residual "core" remains pentameric. This pentameric core fails to enter the nucleus, remaining stably in the cytoplasm. A single tail region attached to the pentamer is sufficient to transport it into the nucleus. The rate of accumulation in the nucleus, but not its final extent, depends on the number of tails per pentamer. The detached (monomeric) tails rapidly accumulate in the oocyte nucleus, indicating that the tail structure is sufficient for selective accumulation. Pentameric cores diffuse throughout the nucleus but are retained when microinjected into the nucleus, indicating that the tail is necessary for entry but not for retention within the nucleus. An improved method for purification of nucleoplasmin is also described.

Amino Acid Sequence↗
PubMed1981

High sequence specificity of micrococcal nuclease.

The substrate specificity of micrococcal nuclease (EC 3.1.4.7.) has been studied. The enzyme recognises features of nucleotide composition, nucleotide sequence and tertiary structure of DNA. Kinetic analysis indicates that the rate of cleavage is 30 times greater at the 5' side of A or T than at G or C. Digestion of end-labelled linear DNA molecules of known sequence revealed that only a limited number of sites are cut, generating a highly specific pattern of fragments. The frequency of cleavage at each site has been determined and it may reflect the poor base overlap in the 5' T-A 3' stack as well as the length of contiguous A and T residues. The same sequence preferences are found when DNA is assembled into nucleosomes. Deoxyribonuclease 1 (EC 3.1.4.5.) recognises many of the same sequence features. Micrococcal nuclease also mimics nuclease S1 selectively cleaving an inverted repeat in supercoiled pBR322. The value of micrococcal nuclease as a "non-specific" enzymatic probe for studying nucleosome phasing is questioned.

Base Sequence↗