Simplified RNA sequencing using dideoxy chain termination.
Explore the source record for details and available documents.
Base Sequence↗
Biomedical subjects
C D Carpenter
Publications and source records attributed to C D Carpenter.
Recombination between satellite RNAs of turnip crinkle virus.
Turnip crinkle virus (TCV) is associated with satellite (sat) RNAs (sat-RNA D, sat-RNA F), defective interfering (DI) RNAs (DI RNA G, DI1 RNA), and one RNA with properties of both sat-RNAs and DI RNAs (sat-RNA C). When plants were inoculated with TCV, sat-RNA D and in vitro sat-RNA C transcripts containing non-viable mutations in the 5' domain, recombinant sat-RNAs were recovered. These recombinants were composed of sat-RNA D at the 5' end and sat-RNA C sequences at the 3' end. Analysis of 20 independent recombination junctions revealed that unequal crossing-over had occurred in planta in a region of sequence similarity between the two sat-RNAs which resulted in the duplication of 3-16 nucleotides. Thirty percent of the sat-RNA recombinants also had one to three additional nucleotides inserted at the crossover junctions which did not correspond to either sat-RNA C or sat-RNA D sequence. The right side of the recombination junctions always began with one of three consecutive nucleotides of sat-RNA C. Based on the similarity between this sequence of sat-RNA C, the right side junction of DI RNA G and the 5' end of TCV, as well as the sequence similarity between right side junctions of DI1 RNA and sat-RNA C and the 5' end of the sat-RNAs, a replicase-driven copy choice mechanism is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Protein kinase C phosphorylation at Thr 654 of the unoccupied EGF receptor and EGF binding regulate functional receptor loss by independent mechanisms.
To test the functional consequence of phosphorylation of the EGF receptor at Thr 654 by protein kinase C, the normal Thr 654 human EGF receptor cDNA or a mutant encoding an Ala 654 were expressed in heterologous cells. In cell lines expressing both the Thr 654 and Ala 654 receptors, functional cell-surface Thr 654 receptors were reduced or were totally lost, but were not degraded, following activation of protein kinase C by phorbol esters (TPA), whereas Ala 654 receptors were unaffected. These data suggest that protein kinase C regulates ligand-independent receptor binding and internalization via phosphorylation of Thr 654 of the EGF holoreceptor. Because EGF induces internalization and degradation of the Ala 654 EGF receptor, at least two independent mechanisms can serve to signal loss of functional EGF receptors.
Alanine↗
Structure of the Spec1 gene encoding a major calcium-binding protein in the embryonic ectoderm of the sea urchin, Strongylocentrotus purpuratus.
We have identified and characterized the structure of the Spec1 gene in the sea urchin Strongylocentrotus purpuratus. In earlier studies we demonstrated that a small family of messenger RNAs, termed Spec mRNAs for S. purpuratus ectodermal mRNAs, begins to accumulate 20 hours after fertilization in ectoderm cells of the sea urchin embryo. The Spec mRNAs code for a group of low molecular weight proteins belonging to the troponin C superfamily. Spec1 transcripts, the predominant mRNAs of the family, are heterogeneous in their 3' untranslated sequences but code for a single protein, recently shown to be a calcium-binding protein. Spec complementary DNA clones were used to isolate genomic clones from two lambda libraries. These genomic clones comprise a 41 kb (kb = 10(3) bases or base-pairs) region of the S. purpuratus genome and contain a Spec1 gene closely linked to another Spec gene, Spec2c. The Spec1 gene is 10.3 kb in length and contains six exons. The genomic clones containing the Spec1 gene can be placed into two groups based on restriction fragment length differences and differences in hybridization strengths using probes derived from Spec1 3' untranslated regions. Evidence that these groups probably correspond to two alleles of the Spec1 gene was obtained by probing genomic DNA blots of sperm DNA from different individuals with 3' untranslated sequences of Spec1 complementary DNA clones. These blots show that two of the Spec1 mRNAs we have characterized, and probably a third, are alleles of the Spec1 gene. Thus, there appears to be a single polymorphic Spec1 gene in the sea urchin genome. We used S1 protection and primer extension procedures to map the 5' end of the Spec1 gene. Results from these experiments indicate that the initiation of transcription of the Spec1 mRNA begins at an A residue 220 bases from the 3' end of the first exon. Adding support to this claim, cannonical T-A-T-A and C-A-A-T sequences, indicative of many eukaryotic promoters, are found 23 bases and 60 bases upstream from this site, respectively. Analysis of sequences within a few kb of the Spec1 gene show that there are five members of a repetitive sequence family near the gene, three upstream and two downstream. The 5' leader sequence of another Spec mRNA, Spec2a, also contains a member of this repeat family.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Novel proteins belonging to the troponin C superfamily are encoded by a set of mRNAs in sea urchin embryos.
The properties of several cDNA clones representing a family of mRNAs found in the embryonic ectoderm of Strongylocentrotus purpuratus are described. We have previously shown that these mRNAs (termed Spec for Strongylocentrotus purpuratus ectoderm) accumulate in the presumptive dorsal ectoderm of post-cleavage stage embryos and code for a group of 10 to 12 low molecular weight acidic proteins. We demonstrate here, using antibodies raised against the major Spec proteins, that the proteins are localized in the cytoplasm of dorsal ectoderm cells. Hybridization analysis and DNA sequencing show that the mRNAs coding for these proteins, although all related, can be divided into two subfamilies. Comparison of the translational reading frames of the Spec mRNAs with known protein sequences shows a significant homology with troponin C-related proteins, especially in the calcium-binding domains. We suggest that the Spec proteins are previously uncharacterized members of the troponin C superfamily.
Animals↗
High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein.
A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli. The vector, designated pMAM17, carries the ColE1 rop gene under the control of the thermally inducible lambda PL promoter. The rop gene product is a negative regulator of ColE1 DNA replication, and its high-level expression is lethal to cells. However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions. pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos. The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily. Large amounts of the Rop-Spec fusion protein were produced at 42 degrees C in E. coli. Unfractionated E. coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos. Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily. This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily.
Animals↗
The 3' untranslated regions of two related mRNAs contain an element highly repeated in the sea urchin genome.
Two closely related cDNA clones, pSpec1 and pSpec2, specifying two developmentally regulated tissue specific mRNAs from sea urchin embryos were used to probe a sea urchin genomic lambda library. Screening 10,000 phage by plaque hybridization yielded several hundred positive signals. With more stringent wash procedures, only two to three phage were positive. Three of these phage, one isolated by stringent wash procedures and two isolated by standard wash procedures were further investigated by restriction analysis, RNA gel blots, and DNA sequencing. The phage isolated by the stringent wash procedure appears to be a gene coding for the Specl mRNA. The other phage contain only partial homology to pSpec1 and pSpec2, 150 to 200 base pairs of the 3' untranslated region of the Spec1 and Spec2 mRNAs. It is concluded that the Spec1 and Spec2 mRNAs contain a highly repetitive element near their 3' end. The element is present at 2000 to 3000 copies per genome and may be transcribed at some sites other than those coding for the Spec1 and Spec2 genes. The possible function and evolutionary origin of the repetitive element is discussed.
Animals↗
A gradient of poly(A)+ RNA sequences in Xenopus laevis eggs and embryos.
Explore the source record for details and available documents.
Animals↗
Open reading frames of turnip crinkle virus involved in satellite symptom expression and incompatibility with Arabidopsis thaliana ecotype Dijon.
Carmoviruses are single-stranded, single component RNA viruses that include turnip crinkle virus (TCV) and the recently discovered cardamine chlorotic fleck virus (CCFV). Full-length, biologically active cDNAs were constructed for the TCV-M isolate and the Blue Lake isolate of CCFV. Using chimeric viruses constructed between isolates of TCV that produce mild or severe symptoms when coinoculated with a virulent satellite RNA, a Glu residue at position 1,144 in the polymerase open reading frame was identified as being involved in satellite-mediated symptom expression. To analyze viral determinants involved in resistance, chimeric viruses with precisely exchanged open reading frames were produced between TCV, which does not infect the Arabidopsis thaliana ecotype Dijon (Di-0), and CCFV, which can infect Di-0, TCV with the coat protein of CCFV was able to systemically infect Di-0 although whole plant hybridizations revealed that the hybrid virus spread more slowly than either of the two parental viruses. These results indicate that the two parental viruses. These results indicate that the coat protein is an important viral determinant in the resistance of Di-0 to TCV.
Amino Acid Sequence↗