Biomedical subjects

D H Giebelhaus

Publications and source records attributed to D H Giebelhaus.

9 recordsLinked to original sources

PubMed1990

Membrane skeleton protein 4.1 in developing Xenopus: expression in postmitotic cells of the retina.

Membrane skeleton protein 4.1 plays a key role in modulating the interactions of spectrin, actin, and integral membrane proteins in erythroid and nonerythroid cells. We have investigated its structure and expression during embryonic development of Xenopus laevis. An analysis of the complete 2758-nucleotide sequence and predicted translation of 801 amino acids (85.5 kDa) of X. laevis oocyte protein 4.1 reveals that, within overlapping regions, oocyte protein 4.1 is 74% identical to a composite amino acid sequence of human erythroid and lymphoid protein 4.1 and has an identity similar to that of amino acid motifs variably expressed in either human erythroid or lymphoid protein 4.1 S1 nuclease protection analysis demonstrates the presence of a single species of protein 4.1 transcript in embryos. Antibodies produced against X. laevis protein 4.1 fusion protein recognize two bands of 180 and 115 kDa on Western blots of X. laevis embryos and retina and, using immunocytochemical techniques, label the developing retina most intensely. In vitro transcription of a cDNA construct fully encoding X. laevis protein 4.1 yields a synthetic mRNA which, when translated in vitro, produces a polypeptide that comigrates on SDS-polyacrylamide gels with the 115-kDa form of embryos and retina. Protein 4.1 is found exclusively in photoreceptors following the terminal mitosis of retinal neurons. When retinal synaptogenesis is complete, protein 4.1 is also expressed in the inner retina. In adult amphibian retinas, protein 4.1 is detected in photoreceptors, bipolar cells, and ganglion cell axons. As these cell types have previously been shown to express spectrin, actin, and ankyrin, it is likely that the membrane skeleton of erythrocytes and retinal cells share functional similarities.

Amino Acid Sequence↗

PubMed1989

Expression of the poly(A)-binding protein during development of Xenopus laevis.

We have isolated and sequenced cDNA clones encoding the poly(A)-binding protein of Xenopus laevis oocytes. Polyclonal antiserum was raised against a fusion protein encoding 185 amino acids of the Xenopus poly(A)-binding protein. This antiserum localizes the poly(A)-binding protein to subcellular sites associated with protein synthesis; in the retina, immunoreactive protein is detected in the synthetically active inner segment of the photoreceptor but not in the transductive outer segment. Transcripts encoding the poly(A)-binding protein are present in oocytes, although no protein is detected on protein blots. In contrast, the levels of both transcripts and protein increase in development, which correlates with the observed increase in total poly(A) during Xenopus embryogenesis (N. Sagata, K. Shiokawa, and K. Yamana, Dev. Biol. 77:431-448, 1980).

Amino Acid Sequence↗

PubMed1988

Antisense RNA inhibits expression of membrane skeleton protein 4.1 during embryonic development of Xenopus.

Plasmids expressing partial-length sense or antisense protein 4.1 RNA were microinjected into fertilized Xenopus eggs. Nuclease protection assays reveal that antisense protein 4.1 RNA lead to the specific loss of endogenous protein 4.1 transcripts after midblastula transition, with no effect on the levels of three unrelated transcripts. As a control, we show that this dramatic loss of endogenous protein 4.1 transcripts is blocked when fertilized eggs receive a second injection of plasmids that express partial-length sense protein 4.1 RNA. Immunocytochemistry of tadpole embryos with antibodies monospecific for protein 4.1 demonstrates that the antisense protein 4.1 RNA blocks the normal expression of protein 4.1 in embryos and interferes with the normal interdigitation of the photoreceptor outer segments with the pigment epithelium layer in the retina. These data suggest that reduced expression of a single membrane skeleton protein is sufficient to perturb normal cellular interactions of the retina.

Amino Acid Sequence↗

PubMed1987

Characterization of murine cDNAs encoding P-57, a neural-specific calmodulin-binding protein.

Polyclonal antibodies raised against bovine brain P-57, a neural-specific calmodulin-binding protein, were used to isolate murine cDNAs encoding P-57 from murine brain cDNA libraries in the expression vector lambda gt 11. Two of the overlapping clones contained an open reading frame encoding a polypeptide of 227 amino acid residues (predicted Mr, 23,635), a 163-nucleotide 5'-untranslated sequence, and a 403-nucleotide 3'-untranslated sequence. Hydrophobicity analysis of the predicted polypeptide indicated the lack of any considerable stretch of hydrophobic residues that may span the membrane. This is consistent with prior data suggesting that P-57 exists in a soluble, as well as a membrane-associated, form. The predicted amino acid composition of P-57 is rather unusual in that it is highly enriched in alanine, glutamic acid, and lysine residues, and relatively enriched with proline residues. This amino acid composition accounts for the very low helical content of the predicted polypeptide. A search of the GenBank and EMBL sequence data banks (GenBank Inc., release 44.0 (August, 1986); European Molecular Biology Library, release 8.0 (April, 1986] indicated that the P-57 nucleotide sequence shows no significant homology to any reported sequences. RNA blot analysis of brain, heart, liver, and testes RNA revealed that cDNAs detect P-57 transcripts of 1.5 kilobases in brain, but not in other tissues. Genome blot analysis was consistent with P-57 being encoded by a single or small number of genes. These data demonstrate that the accumulation of this novel calmodulin-binding polypeptide in neural tissue is controlled primarily at the level of RNA abundance.

Amino Acid Sequence↗

PubMed1987

Changes in the expression of alpha-fodrin during embryonic development of Xenopus laevis.

Fodrin (nonerythroid spectrin) and its associated proteins have been previously implicated in the establishment of specialized membrane-cytoskeletal domains in differentiating cells. Using antiserum which is monospecific for the alpha-subunit of fodrin, we demonstrate that alpha-fodrin is present in oocytes and adult tissues of Xenopus laevis. Analyses of the de novo synthesis of alpha-fodrin during embryonic development reveal that alpha-fodrin is synthesized in oocytes, but not during early development. To investigate the level of control of alpha-fodrin expression, we isolated two cDNA clones for oocyte alpha-fodrin. The oocyte cDNA clones were identified as encoding portions of alpha-fodrin based on DNA sequence analysis and on the comparison of the predicted amino acid sequence of the cDNAs with the known sequence of human erythrocyte alpha-spectrin. The Xenopus alpha-fodrin cDNAs hybridize to a transcript of approximately 9 kb on RNA blots, and probably to a single gene type on genomic DNA blots. Both RNA blot analyses and S1 nuclease protection assays with the Xenopus alpha-fodrin cDNAs demonstrate that the observed decline in the de novo synthesis of alpha-fodrin polypeptides is controlled by a dramatic decrease in the abundance of alpha-fodrin transcripts after fertilization. In contrast, levels of actin transcripts do not decrease during this period. Inasmuch as steady-state levels of alpha-fodrin transcripts rise by the neurula stage of development, these results suggest that the synthesis of alpha-fodrin polypeptides during embryonic development of Xenopus is regulated, rather than constitutive, and that the primary level of control is the steady-state abundance of mRNA.

Amino Acid Sequence↗

PubMed1987

cDNA cloning, sequencing and chromosome mapping of a non-erythroid spectrin, human alpha-fodrin.

Several overlapping cDNA clones encompassing 2760 nucleotides of the alpha-subunit of a human non-erythroid spectrin (termed fodrin) were isolated from a human lung fibroblast cDNA library. DNA and RNA blot analyses indicated that a single copy alpha-fodrin gene encodes a 9-kb transcript. The cDNA clones were sequenced, and all were found to contain long open reading frames. The overlapping regions were identical except for a 60-nucleotide inframe insertion at position 1133 in the composite sequence. This result suggests that at least two distinct transcripts exist in fibroblast cells. The chromosomal location of human alpha-fodrin was assigned to 1p34-1p36.1 by hybridization to somatic cell hybrids, and it is thus distinct from that of human alpha-spectrin which has been mapped to 1q22-1q25. Alignment of the composite 919 amino acids of the predicted protein sequence of human alpha-fodrin with that of human alpha-spectrin indicated that alpha-fodrin has a similar 106-amino-acid repeating structure, which is homologous with alpha-spectrin repeats 7-15. Repeats 10 and 11 are anomalous in sequence and structure from other repeats. A comparison of nucleic acid and amino acid homologies between alpha-spectrin and the alpha-fodrin of several vertebrates indicated that human non-erythroid alpha-fodrin and the common alpha-subunit of erythroid and non-erythroid cells of non-mammalian vertebrates are closely related (90%-96% amino acid homology), whereas alpha-fodrin is only distantly related to the erythroid-specific alpha-spectrin subunit of mammals (55%-59% amino acid homology). These data suggest that mammalian erythroid alpha-spectrin evolved by duplication and rapid divergence from an ancestral alpha-fodrin-like gene.

Amino Acid Sequence↗

PubMed1985

Actin mRNA content in normal and delayed implanting mouse embryos.

Actin mRNA levels were measured in mouse eggs, early embryos, and delayed implanting blastocysts by a homologous, cloned recombinant DNA probe and "dot" blot methodology. A maternal store of 431 fg of actin mRNA was observed in the unfertilized eggs. This mRNA pool decreased 12-fold by the mid-two-cell stage. Actin mRNA levels were then observed to increase progressively from the eight-cell to the blastocyst stage on a basis proportional to cell number. Late blastocysts contained 2400 fg actin mRNA per embryo (22 fg per cell). The cellular level decreased by about 20% in embryos induced into delay of implantation by ovariectomy of donor females. Reactivation of the delayed implanting blastocysts through hormonal manipulation in vivo or culture in vitro was accompanied by reestablishment of the level of cellular actin mRNA observed in normal blastocysts.

Actins↗

PubMed1985

Quantitative and qualitative changes in histone gene expression during early mouse embryo development.

There are large amounts of histone mRNA present in mouse eggs. These RNAs are rapidly degraded, as are other mRNAs, after fertilization and prior to the second cleavage. During cleavage, the histone mRNA accumulates as the embryo divides. The same sets of histone genes are expressed in eggs and embryos, although there are large qualitative differences in the amounts of particular histone mRNAs. The function of the egg histone mRNA is unknown. The amount of histone mRNA in cleaving and blastocyst embryos is probably sufficient to code for the blastocyst histone proteins.

Animals↗

PubMed1983

Changes in the quantity of histone and actin messenger RNA during the development of preimplantation mouse embryos.

Actin and histone H3 mRNA levels in mouse eggs and early embryos have been measured by use of recombinant DNA probes having sequence homology to those mRNA species. Total nucleic acid was extracted from pools of unfertilized eggs, two-cell embryos, eight-cell embryos, and blastocysts. The nucleic acids were resolved electrophoretically, bound to diazotized paper following Northern transfer, and hybridized with 32P-labeled histone or actin DNA probes. Our findings demonstrate that there is a maternal store of histone and actin mRNA in the unfertilized egg but that this mRNA pool is reduced roughly 10-fold on an embryo basis by the mid-two-cell stage. Following this reduction of maternal mRNA, histone and actin mRNA accumulation from the eight-cell cleavage stage to the blastocyst increases proportionally to cell number and appears to be controlled by zygote genome transcription.

Actins↗