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L D Etkin

Publications and source records attributed to L D Etkin.

At least 19 recordsLinked to original sources
PubMed1992

Xlcaax-1 is localized to the basolateral membrane of kidney tubule and other polarized epithelia during Xenopus development.

Xlcaax-1 is a novel, maternally expressed, 110-kDa, CAAX box containing protein that undergoes isoprenylation and palmitoylation through which it associates with the plasma membrane. We report here the cellular and subcellular localization of the xlcaax-1 protein during development of Xenopus laevis. Whole-mount immunocytochemistry and immunoperoxidase staining of tissue sections show that during development the xlcaax-1 protein accumulation is coincident with the differentiation of the epidermis, pronephros, and mesonephros. In the pronephros and mesonephros the xlcaax-1 protein is localized to the basolateral membrane of differentiated tubule epithelial cells. Thus, the xlcaax-1 protein serves as a marker for tubule formation and polarization during Xenopus kidney development. Xlcaax-1 may also be used as a marker for the functional differentiation of the epidermis and the epidermally derived portions of the lens and some cranial nerves. Western blot analysis shows that in the adult the xlcaax-1 protein is most abundant in kidney. Immunogold EM analysis shows that the xlcaax-1 protein is highly enriched in the basal infoldings of the basolateral membrane of the epithelial cells in adult kidney distal tubules. In addition, immunoperoxidase staining of tissue sections detected low levels of xlcaax-1 protein in the epithelial cells of skin, urinary bladder, gall bladder, and parietal glands of the stomach. The localization pattern of xlcaax-1 suggests that the protein may function in association with an ion transport channel or pump.

Animals↗
PubMed1992

The cloning and characterization of a localized maternal transcript in Xenopus laevis whose zygotic counterpart is detected in the CNS.

We have cloned a cDNA (xlan4) from a Xenopus laevis oocyte cDNA library whose cognate mRNA is localized in the animal pole region of full grown oocytes. The cDNA can be translated in vitro to produce a predicted size protein of 35 kDa and, is also expressed in E. coli as a fusion protein. The conceptual protein encoded by the xlan4 cDNA is 17.5% proline rich and possesses several PEST sequences found in proteins with short half-lives. The xlan4 mRNA is 2.6 kb and during early development its titer decreases until the neurula stage after which it begins to reaccumulate. Northern blots on dissected embryos and in situ hybridization revealed that the zygotic expression is limited to the dorsal axial structures consisting primarily of the CNS. UV irradiation of the vegetal pole region immediately following fertilization that produces ventralized embryos results in a loss of zygotic xlan4 expression. In the adult, xlan4 mRNA is limited primarily to the brain. The presence of this mRNA in animal pole region which contributes to the future neural cell lineages suggests that this gene product may function either in the specification of neural cell types or in a neural specific function.

Amino Acid Sequence↗
PubMed1991

Identification of the cDNA for xlcaax-1, a membrane associated Xenopus maternal protein.

xlcaax-1 is a cDNA coding for a CAAX box containing protein in Xenopus laevis that undergoes isoprenylation and palmitoylation. Here we report on the confirmation that this clone (formerly xlgv7) codes for a 110 kDa membrane associated protein and not an 80 kDa nuclear protein as originally believed (1). The reason for the misidentification was the presence of a common epitope on these two proteins recognized by the monoclonal antibody 37-1A9. We clarified the discrepancy by raising polyclonal antibodies against the xlcaax-1 protein produced in a bacterial expression system and demonstrating that these antibodies only recognize the 110 kDa protein on western blots of oocyte extracts. During early development xlcaax-1 protein starts reaccumulating from the neurula stage. In the adult frog both the xlcaax-1 protein and its cognate mRNA are highly enriched in the kidney. Consistent with the presence of CAAX box at the C-terminus this protein is associated with the membranes in Xenopus tissue culture cells (XTC).

Animals↗
PubMed1991

A novel 110-kDa maternal CAAX box-containing protein from Xenopus is palmitoylated and isoprenylated when expressed in baculovirus.

We describe a unique 110-kDa protein, xlcaax-1, that is a member of a group of membrane-associated proteins such as the ras and ras-related proteins and nuclear lamins. Many of these proteins are involved in signal transduction or cell signaling, possess a C-terminal CAAX box, and undergo fatty acid acylation (Glomset, J. A., Gelb, M. H., and Farnsworth, C. C. (1990) Trends Biochem. Sci. 15, 139-142). The ras and ras-related proteins bind GTP and in most cases are both isoprenylated and palmitoylated. The xlcaax-1 protein possesses a C-terminal CAAX sequence that is identical to the N-ras protein. In addition to the CAAX box, xlcaax-1 contains a series of basic amino acids upstream of the CAAX sequence similar to several nonpalmitoylated forms of the ras-related proteins. When the xlcaax-1 cDNA is expressed in a baculovirus expression system, the product undergoes isoprenylation and palmitoylation utilizing a mechanism similar to that of the ras proteins. In addition, the xlcaax-1 protein is isoprenylated, and a minor fraction is palmitoylated in Xenopus XTC tissue culture cells. We have also demonstrated that the protein is associated with membrane fractions in full-grown Xenopus oocytes and in Xenopus XTC tissue culture cells and that membrane association is isoprenylation-dependent. The presence of maternal molecules possessing signal transduction potential is an attractive mechanism for modulating the effects of growth factors and other signal molecules during development.

Amino Acid Sequence↗
PubMed1991

x121: a localized maternal transcript in Xenopus laevis.

We describe the cloning and characterization of a partial cDNA, x121, that represents an RNA, which is localized in the animal hemisphere of Xenopus oocytes. This RNA is also detected in an animal to vegetal gradient during early cleavage stages. The x121 RNA titer decreases from fertilization through the gastrula stage, after which it is not detectable on northern blots. The amino acid composition of the x121 conceptual protein derived from cDNA sequencing reveals a large number of acidic residues similar in distribution to proteins that function as transcriptional activators.

Amino Acid Sequence↗
PubMed1991

The nuclear-cytoplasmic distribution of the Xenopus nuclear factor, xnf7, coincides with its state of phosphorylation during early development.

We describe the characterization in Xenopus laevis of a nuclear protein, xnf7, which is first detected in the oocyte GV and is eventually enriched in nuclei of cells of the adult brain. Previous studies have shown that this protein contains zinc-finger-like structures and acidic domains typical of transcriptional activators, and is phosphorylated in vitro by p34cdc2 protein kinase. The protein also binds to double-stranded DNA. These data suggest that xnf7 may function as a transcription factor. During oocyte maturation, xnf7 is released into the cytoplasm and is not detectable in nuclei until the mid-blastula-gastrula stage of development. Western blot analysis of xnf7 isolated from oocytes and eggs showed the existence of multiple bands or isoforms of the protein. Unique isoforms that are generated during oocyte maturation are the result of phosphorylation. The phosphorylated isoforms remain in the cytoplasm until the mid-blastula stage. The re-accumulation of protein in the embryonic nuclei at this time correlates with the increase in abundance of the less phosphorylated isoforms. The xnf7 protein possesses a nuclear localization signal (NLS) similar to the bipartite signal found in nucleoplasmin. Newly synthesized xnf7 accumulated in the oocyte GV to detectable levels within a few hours following synthesis suggesting that retention of the protein in the cytoplasm during early cleavage may be due to a process that interferes with the function of the NLS. These data suggest that compartmentalization and/or post-translational modification of the nuclear protein xnf7 may be involved in regulating its function during early development.

Animals↗
PubMed1988

Regulation of the mid-blastula transition in amphibians.

The cleavage cycles during early amphibian development are synchronous, rapid, and biphasic. There is no transcription and no growth of the embryo, and the nuclear cycle is independent of the cytoplasmic cleavage cycle. All components necessary for development through the blastula stage are provided by maternal stores. At the twelfth cleavage division, a major transition occurs that involves initiation of transcription, an elongation of the cell cycle, an increase in cell movement, and asynchrony of cell division. It is probable that the major controlling factor in the regulation of all the aforementioned events is the cell cycle. During early cleavage stages, the cell cycle is both rapid (30-35 min) and synchronous. There is evidence that the cycling time may be controlled by the presence of several mitotic factors, such as MPF, CSF (cytostatic factor), and a titratable component that binds to nuclear membranes. The rapid rate of DNA synthesis may inhibit the formation of transcription complexes, resulting in the absence of detectable transcription before the MBT. Cellular movement may also be inhibited in the rapidly dividing cell. As the cell cycle elongates (possibly due to the functional loss or sequestration of one or more of the mitotic control factors), the G1 and G2 phases are incorporated into the cell cycle. Under conditions of slower rates of DNA replication and the presence of the G1 and G2 phases, the transcriptional machinery becomes functional. It is apparent that at the MBT, not all classes of transcripts are activated simultaneously in every cell, nor is their expression regulated by a common mechanism. Incorporation of the G1 and G2 phases in the cell cycle may also permit the synthesis and assembly of microtubules and cytoskeletal components necessary for the initiation of the cell movements characteristic of this stage of development. The role of the cell cycle in controlling events at the MBT is supported by evidence from studies in which perturbation of the cell cycle that results in its elongation or arrest produces subsequent initiation of events that occur normally at the MBT. The MBT therefore appears to be a window in the developmental time frame, during which a number of molecular and morphogenetic events occur independently of one another, but all are necessary for subsequent morphogenesis and cellular differentiation. It is a transition from the strict reliance on the maternal program to a dependence on the new transcription from the embryonic genetic program. It is probable that the major regulatory mechanism involved in the occurrence of this constellation of cellular events is the change in the cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Persistence and replication of plasmid DNA microinjected into early embryos of Xenopus laevis.

The persistence and replication of defined circular and linear plasmid DNA molecules microinjected into fertilized eggs of Xenopus laevis were analyzed. For all plasmids tested, a small fraction of microinjected circular molecules was replicated; however, the overall copy numbers of either free form I or form II molecules usually did not increase through blastulation. In contrast, extensive amplification of input DNA sequences was seen whenever the microinjected DNA was assembled into high molecular weight concatemers. Moreover, the appearance and subsequent replication of injected sequences in high molecular weight DNA were enhanced when linear (form III), rather than circular, molecules were microinjected. The injected form III DNA was rapidly converted into long linear concatemers. All possible orientations of monomeric molecules within the concatemers were observed although, on occasion, head-to-tail orientations were favored. Long linear concatemers were replicated very efficiently, irrespective of the sequence of the input DNA. Form I and form II DNA molecules were also formed in the embryo from microinjected form III DNA. A small fraction of these circular forms was replicated, although overall copy numbers did not increase significantly. Form III molecules that remained monomeric were not observed to be replicated at all within our limits of detection. In some batches of embryos, form I and form II DNA molecules were replicated to the extent that overall copy number increased. Even in these cases, however, the amplification of long linear concatemers of the input DNA sequences was more efficient.

Animals↗
PubMed1988

Competition between DNA templates injected into Xenopus laevis oocytes.

We have examined the competition between microinjected DNA templates for endogenous Xenopus oocyte transcription factors. The results suggest that (a) coinjected templates compete with one another for transcription factors, (b) the competitive ability of a template is not necessarily a function of the rate of transcription of the gene, (c) competition is due to the presence of specific DNA sequences, and (d) preincubation of the test gene affects competition.

Animals↗
PubMed1987

Replication of injected DNA templates in Xenopus embryos.

We have analysed the replication of both exogenous frog DNAs and heterologous DNAs during development from the first cleavage through the blastula stage, by their microinjection into fertilized eggs of Xenopus laevis. The data show that various plasmids increase to different extents and that the differences cannot be attributed to size alone. Plasmids containing the Xenopus ribosomal gene repeat unit do not replicate efficiently, and they also inhibit the replication of co-injected DNA templates. This inhibitory effect may be due to DNA sequences contained in the intergenic ribosomal gene spacers.

Animals↗
PubMed1987

Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs.

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

Acetyltransferases↗
PubMed1985

Secretion of ceruloplasmin by a human clear cell carcinoma maintained in nude mice.

Ceruloplasmin is the best known but least understood copper protein. Studies preliminary to investigating the control of ceruloplasmin synthesis have utilized a human renal cell carcinoma maintained in nude mice for 73 passages over a 5-year period. In vitro cultures of these cells were accomplished and the mRNAs were extracted prior to microinjection into Xenopus oocytes. The media examined by SE-HPLC and immunological techniques demonstrated that (1) after in vitro culture, ceruloplasmin was secreted as an uncleaved polypeptide chain with a MW of 135,000; (2) the translational product of ceruloplasmin mRNA injected into Xenopus oocytes was cleaved into fragments with MWs of 110,000, 67,000, and 50,000. The results indicate that mRNA for human ceruloplasmin can be obtained to serve as a template for the synthesis of a cDNA probe to investigate the control of human ceruloplasmin's synthesis.

Adenocarcinoma↗
PubMed1985

Transformed Xenopus embryos as a transient expression system to analyze gene expression at the midblastula transition.

The onset of transcriptional activity during embryogenesis in Xenopus laevis is at the 4000- to 8000-cell stage (stage 8-8.5) and is referred to as the midblastula transition (MBT). Most exogenous circular DNA that is microinjected into the fertilized egg also is expressed at the MBT. The transformed Xenopus embryo at these early stages was used as a transient expression system in order to determine the effects of (1) promoter strength, (2) physical conformation, (3) degree of replication, and (4) a regulatory molecule on the expression of an injected gene coding for chloramphenicol acetyl transferase. This gene linked to a relatively strong promoter (SV40 early promoter -pSV2CAT), a weak promoter (adenovirus early promoter -pE3CAT), or in circular or linear form is expressed at stage 8-8.5 following injection into fertilized eggs. pE3CAT coinjected with the E1a protein (enhances the transcription of the E3 promoter) is also expressed at stage 8.5, but expression is enhanced 2-7.6 fold. These data suggest that the inhibition of transcription prior to the MBT could not be perturbed by either the presence of different promoters or a positive regulatory molecule such as the E1a protein.

Acetyltransferases↗