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PubMed1995

An abundant, trans-spliced mRNA from Toxocara canis infective larvae encodes a 26-kDa protein with homology to phosphatidylethanolamine-binding proteins.

A full-length mRNA encoding a secreted 26-kDa antigen of infective larvae of the ascarid nematode parasite Toxocara canis has been identified. This was characterized as a 1,082-base pair clone highly abundant (0.8-1.9%) in cDNA prepared from infective stage larvae but absent from cDNA from adult male worms. Sequence analysis revealed an open reading frame corresponding to a hydrophilic 263-amino acid residue polypeptide with a 20-residue N-terminal signal peptide, indicating that it is secreted. The 5' end of the cDNA was isolated by polymerase chain reaction using a primer containing the nematode-spliced leader sequence, SL1, showing that the mRNA is trans-spliced. The molecular mass of the putative protein with the signal peptide removed is 26.01 kDa, and antibody to the recombinant protein expressed in bacterial vectors reacts with a similarly sized protein in T. canis excretory/secretory (TES) products. An identical sequence was obtained from a genomic clone isolated by expression screening with mouse antibody to TES. The 72 amino acid residues adjacent to the signal peptide form two homologous 36-residue motifs containing 6 cysteine residues; this motif is found also in the T. canis-secreted glycoprotein TES-120 and in genes of Caenorhabditis elegans. Sequence data base searches revealed significant similarity to 7 other sequences in a newly recognized gene family of phosphatidylethanolamine-binding proteins that includes yeast, Drosophila, rat, bovine, simian, and human genes and a representative from the filarial nematode Onchocerca volvulus. Assays with the T. canis recombinant 26-kDa protein expressed as a fusion with maltose-binding protein have confirmed phosphatidylethanolamine-binding specificity for this novel product.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of TPAR1, a gene whose expression is repressed by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA).

We previously isolated a partial cDNA sequence, termed TPAR1 (TPA repressed gene 1), from a cDNA library constructed from C3H10T1/2 mouse embryo fibroblasts treated with TPA, using a differential screening procedure. (M.D. Johnson et al. Mol. Cell. Biol. 7, 2821-2829, 1987). In the present study, we have cloned two corresponding full-length 1.9- and 3.4-kb cDNAs of TPAR1 from murine cDNA libraries. Sequence analysis of these TPAR1 cDNAs revealed that they encode 89 and 93 amino acid polypeptides, respectively, with a putative leader sequence and show significant homology with the human cytokine interleukin-8 (IL-8) and its superfamily. Genomic DNA isolation and structural characterization provide evidence that the TPAR1 mRNAs are transcribed from a single gene with alternative splicing. TPAR1 mRNAs are expressed ubiquitously among adult mouse tissues as three major transcripts, 1.9, 3.4, and 6.5 kb, whose expression depends on the tissue type. The levels of TPAR1 mRNAs were markedly decreased in fibroblasts following TPA treatment and also in serum-deprived quiescent fibroblasts stimulated by serum. The levels of TPAR1 mRNAs were dramatically down-regulated in regenerating rat liver when compared to normal adult liver. In addition, there was no detectable expression of TPAR1 in three rat hepatoma cell lines and several transformed fibroblast cell lines. Thus, the TPAR1 gene is a new member of the cytokine IL-8 superfamily, whose expression is down-regulated in rapidly dividing cells. Further studies are required to determine whether it plays a negative role in controlling cell proliferation and tumorigenesis.

Amino Acid Sequence↗
PubMed1994

T cell receptor gamma cDNA in human fetal liver and thymus: variable regions of gamma chains are restricted to V gamma I or V9, due to the absence of splicing of the V10 and V11 leader intron.

Although complete in-frame transcripts of the human T cell receptor gamma V10 and V11 genes have been described, the corresponding gamma chains have never been found in gamma delta T cell receptors. In this study, we show that the leader intron of all V10 and V11 cDNA isolated from fetal thymus, fetal liver and adult peripheral blood lymphocytes are unspliced. We demonstrate that, due to the absence of splicing, V10 and V11 are pseudogenes and cannot be expressed in gamma chains. They are the first pseudogenes of this type described in a rearranging T cell receptor/immunoglobulin locus. Therefore the gamma repertoire at the protein level is limited to subgroup V gamma I and to V9. By analysis of the gamma polymerase chain reaction products from total cDNA, we find that the gamma locus is active in early ontogeny (8 weeks), as shown by the presence of rearranged V9 and V10 gene transcripts in the liver. At 13 weeks, the V gamma I genes as well as V9 and V10 have undergone productive rearrangements in the liver, and in the thymus. Most rearrangements, if not all, involve the T cell receptor gamma C1 region (JP1, JP, J1 segments) in both tissues, confirming the accessibility of the C1 region in early stages of development.

Amino Acid Sequence↗
PubMed1998

Coamplification and dispersion of adjacent human endogenous retroviral HERV-H and HERV-E elements; presence of spliced hybrid transcripts in normal leukocytes.

In an RT-PCR study of HERV-H spliced subgenomic transcripts, we found transcripts with HERV-H leader and protease-encoding sequences spliced to HERV-E integrase-encoding sequences in lymphocytes from healthy blood donors. In other cell types, including two T-cell leukemia cell lines, these transcripts were absent. The PCR fragments of the hybrid transcripts contained two open reading frames (ORFs). One was a hybrid HERV-H protease/HERV-E integrase ORF and the other was the HERV-E envelope surface glycoprotein ORF. Alternative splice products were also identified. The genomic DNA origin of the hybrid transcripts was shown to be a HERV-H element with a large 3'-end deletion, adjacent to a HERV-E element lacking the 5'-LTR. This hybrid structure was shown to be amplified and dispersed to six different human chromosomes. Thus, a relatively large part of full-length HERV-E elements (15-20%) is potentially under the transcriptional control of HERV-H LTRs. The HERV-H/HERV-E junction was present in multiple copies also in the chimpanzee and gorilla, but not in the orangutan or old world monkeys.

Alternative Splicing↗
PubMed1994

Origin, properties, and regulated expression of multiple mRNAs encoded by the protein kinase C1 gene of Caenorhabditis elegans.

Recently, we cloned and characterized cDNA encoding a novel, protein kinase C (designated PKC1B) from Caenorhabditis elegans. PKC1B (707 amino acid residues) is a developmentally regulated, calcium-independent kinase that is expressed exclusively in sensory neurons and related interneurons. We have now discovered a mechanism by which a second, distinct mRNA (PKC1A mRNA) with increased protein coding potential is generated from the C. elegans PKC1 gene. PKC1A mRNA is produced in a process that involves the utilization of an alternative, distal promoter, the incorporation of two unique exons into the mRNA, and alternative cis/trans splicing. Diversity among PKC1 gene transcripts is increased substantially by trans-splicing. The 5' end of PKC1A mRNA contains an acceptor site that is modified by the addition of either a classical spliced leader sequence 2 or one of four novel spliced leaders. PKC1A mRNA encodes a predicted kinase that contains the entire sequence of PKC1B as well as an N-terminal extension of 56 residues. The extension contains a preponderance of basic amino acids. The levels of transcripts arising from the distal (1A) and proximal (1B) promoters for the PKC1 gene are differentially regulated during C. elegans development. The ratio of 1B mRNA:1A mRNA varies from 40:1 to unity as the nematodes progress from early larval stages to mature adults. The novel exons in the PKC1A structural gene are not contiguous with the PKC1A promoter but are instead positioned downstream from a second gene, kinase upstream gene-1, in the context of a multicystronic operon. PKC1A and kinase upstream gene-1 mRNAs are coordinately expressed in a fixed ratio throughout C. elegans post-embryonic development, suggesting that a shared upstream promoter regulates transcription of both genes. Finally, PKC1A and PKC1B mRNA levels are differentially regulated by phorbol esters in a process that may involve the participation of another PKC isoform that is analogous to mammalian PKC delta.

Amino Acid Sequence↗
PubMed1982

Analysis by microinjection of the biological effects of site-directed mutagenesis in cloned avian leukosis viral DNAs.

Cloned avian leukosis viral DNAs were mutagenized in the long terminal repeat, in the leader sequence for env mRNA, and at the poly-env junction. The effect of these mutations in the viral DNA upon its ability to direct virus production or env mRNA synthesis was analyzed by microinjecting the mutant DNAs into chicken embryo fibroblasts and into chicken cells transformed by the env-deficient Bryan strain of Rous sarcoma virus, respectively. The results indicated that: (i) addition of up to 8 base pairs 19 nucleotides upstream of the Hogness box did not block transcription; (ii) deletion of 26 base pairs, including the tRNA primer binding site, allowed synthesis of all viral products and participation in recombination, but replication was blocked; (iii) deletion of fewer than 50 base pairs 250 bases downstream of the long terminal repeat depressed expression of all viral genes; and (iv) deletion of most of the gag and pol genes did not inhibit env mRNA synthesis, but virion packaging of the unspliced transcript was inefficient.

Animals↗
PubMed1983

Detailed analysis of the portion of the herpes simplex virus type 1 genome encoding glycoprotein C.

We previously showed that the right third of HindIII fragment L (0.59 to 0.65) of herpes simplex virus type 1 (HSV-1) encodes a family of mRNAs some members of which appear to be related by splicing. In the experiments described in this communication, we determined the nucleotide sequence of the DNA encoding this mRNA family and precisely located the mRNAs associated with this DNA sequence. The major mRNA species is unspliced and encoded by a 2.520-nucleotide region. Just upstream of the 5' end are TATA and CAT box sequences characteristic of HSV-1 promoters. The 3' end maps near a region containing a nominal polyadenylation signal. Three minor species (2,400, 2,200, and 1,900 bases, respectively) appear to share a very short leader sequence with the 5' end of the major mRNA and are then encoded by uninterrupted DNA sequences beginning about 100, 400, and 625 bases downstream of the 5' end of the major unspliced mRNA. These positions map at or very near positions which agree reasonably well with consensus splice acceptor sequences. The fourth mRNA is encoded by a contiguous 730-nucleotide sequence at the 3' end of the major unspliced mRNA and has its 5' end just downstream of recognizable TATA and CAT box sequences. We suggest that this mRNA is controlled by its own promoter. The nucleotide sequence data, in combination with the mRNA localization, demonstrate four potential polypeptides encoded by the region. The largest is 1,569 bases long and defines a 523-amino acid protein with sequence features characteristic of a glycoprotein. This was confirmed to be HSV-1 glycoprotein C by immune precipitation of the in vitro translation product of the major unspliced mRNA, performed with a polyspecific antibody to HSV-1 envelope glycoproteins (anti-env-1 serum), and by comparison of tryptic peptides of this translation product with those of authentic HSV-1 glycoprotein C. Polypeptides encoded by some of the minor species also were tentatively identified.

Base Sequence↗
PubMed1998

cDNA sequencing and analysis of POV1 (PB39): a novel gene up-regulated in prostate cancer.

We recently identified a novel gene (PB39) (HGMW-approved symbol POV1) whose expression is up-regulated in human prostate cancer using tissue microdissection-based differential display analysis. In the present study we report the full-length sequencing of PB39 cDNA, genomic localization of the PB39 gene, and genomic sequence of the mouse homologue. The full-length human cDNA is 2317 nucleotides in length and contains an open reading frame of 559 amino acids which does not show homology with any reported human genes. The N-terminus contains charged amino acids and a helical loop pattern suggestive of an srp leader sequence for a secreted protein. Fluorescence in situ hybridization using PB39 cDNA as probe mapped the gene to chromosome 11p11.1-p11.2. Comparison of PB39 cDNA sequence with murine sequence available in the public database identified a region of previously sequenced mouse genomic DNA showing 67% amino acid sequence homology with human PB39. Based on alignment and comparison to the human cDNA the mouse genomic sequence suggests there are at least 14 exons in the mouse gene spread over approximately 100 kb of genomic sequence. Further analysis of PB39 expression in human tissues shows the presence of a unique splice variant mRNA that appears to be primarily associated with fetal tissues and tumors. Interestingly, the unique splice variant appears in prostatic intraepithelial neoplasia, a microscopic precursor lesion of prostate cancer. The current data support the hypothesis that PB39 plays a role in the development of human prostate cancer and will be useful in the analysis of the gene product in further human and murine studies.

Amino Acid Sequence↗
PubMed1996

Complete structure of the human alpha-albumin gene, a new member of the serum albumin multigene family.

The nucleotide sequence of the human alpha-albumin gene, including 887 bp of the 5'-flanking region and 1311 bp of the 3-flanking region (24,454 in total), was determined from three overlapping lambda phage clones. The sequence spans 22,256 bp from the cap site to the polyadenylylation site, revealing a gene structure of 15 exons separated by 14 introns. The methionine initiation codon ATG is within exon 1; the termination codon TGA is within exon 14. Exon 15 is entirely untranslated and contains the polyadenylylation signal AATAAA. The deduced polypeptide chain is composed of a 21-amino-acid leader peptide, followed by 578 amino acids of the mature protein. There are seven repetitive DNA elements (Alu and Kpn) in the introns and 3-flanking region. The sizes of the 15 alpha-albumin exons match closely those of the albumin, alpha-fetoprotein, and vitamin D-binding protein genes. The exons are symmetrically placed within the three domains of the individual proteins, and they share a characteristic codon splitting pattern that is conserved among members of the gene family. The results provide strong evidence that alpha-albumin belongs to, and most likely completes with, the serum albumin gene family. Based on structural similarity, alpha-albumin appears to be most closely related to alpha-fetoprotein. The complete structure of this family of four tandemly linked genes provides a well-characterized approximately 200 kb locus in the 4q subcentromeric region of the human genome.

Albumins↗
PubMed2002

A long leader intron of the Ostub16 rice beta-tubulin gene is required for high-level gene expression and can autonomously promote transcription both in vivo and in vitro.

A 2 kb DNA fragment, upstream of the rice beta-tubulin isotype 16 (Ostub16) coding sequence, was isolated using inverse PCR and screening of a tubulin-enriched lambda library. An intron (863 bp) present in the 5' untranslated region (5' UTR) is spliced out to produce the most abundant mRNA species which corresponds to the previously cloned Ostub16 cDNA. Transient expression assays performed on rice embryogenic calluses with chimeric Ostub16::GUS constructs demonstrated that the entire 2 kb upstream sequence has a strong promoter activity, and that the 863 bp intron is required for high-level GUS expression. In addition, the intron sequence is capable per se of sustaining a weak but consistent GUS expression. Two rare Ostub16 transcripts, with a start site mapping within this intron sequence, were detected in rice coleoptile cells. The transcription start site mapped at position -290 with respect to the ATG codon, and the shorter molecule originated from splicing of the same precursor mRNA. Therefore transcriptional expression of rice beta-tubulin isotype 16 results in the synthesis of two premRNA molecules (I and II) encoding for three different mRNA species. We discuss these findings in terms of function and molecular evolution of the mechanisms that control plant beta-tubulin gene expression.

5' Flanking Region↗
PubMed1994

Insulin-like growth factor-I gene expression in human granulosa-lutein cells.

IGF-I is an important local regulator of ovarian function, stimulating follicular growth and steroidogenesis in human granulosa cells. However, it is not known whether ovarian IGF-I is derived from the circulating serum pool or from local production. IGF-I peptide has only been detected in human thecal cells and not in granulosa cells. This study has used the sensitive technique of reverse transcription of mRNA followed by PCR amplification (RT/PCR) to examine IGF-I gene expression in human preovulatory granulosa cells. Granulosa-lutein cell (GLC) samples were obtained by follicular puncture of seven women enrolled in an ovulation induction programme. Treatment had included buserelin acetate, human menopausal gonadotrophin to stimulate follicular growth and human chorionic gonadotrophin to induce ovulation. Total RNA (TRNA), extracted from the GLCs, was amplified by RT/PCR, using combinations of leader and 3' IGF-I exon-specific primers, to yield four IGF-I gene products: IGF-IA (exons 1, 3, 4, 6), IGF-IB (exons 1, 3, 4, 5), IGF-IA' (exons 2, 3, 4, 6) and IGF-IB' (exons 2, 3, 4, 5). As controls from other tissues, an identical procedure was undertaken on TRNA from peripheral blood monocytes and liver. All four mRNAs were expressed in GLCs, monocytes and liver. However the pattern of IGF-I mRNA expression differed between the tissues; in liver and GLCs, the IGF-IA transcript was dominant, but in monocytes the IGF-IA' species was the most prominent. Quantitative RT/PCR using standardization to the house-keeping gene for glyceraldehyde-3'-phosphate dehydrogenase revealed that IGF-IA mRNA was 300-fold more abundant in liver than GLCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Isolation and characterization of a cDNA for osteopontin-k: a kidney cell adhesion molecule with high homology to osteopontins.

Screening of a bovine renal cDNA library with MAbs resulted in the isolation of a 1447 bp cDNA. This cDNA (pBk2.1) was sequenced and shown to contain an open reading frame with a putative protein of 261 amino acids, with a molecular weight of 29,573 (minute leader sequence) and a hydrophobic leader sequence of 16 amino acids. pBk2.1 was shown to share a high level of nucleic acid sequence homology over portions of its sequence to human, porcine, mouse, and rat osteopontins (40-60%). The peptide (osteopontin-k) had a potential glycosylation site (Asn-X-Ser/Thr), a GRGDS receptor binding region, a high level of asparagine residues, and a high abundance of acid amino acids characteristic of osteopontin-like cell adhesion molecules. The N-terminal amino acid region of pBk2.1 (the first 82 amino acids) and 42 amino acids at the C terminus had the highest level of homology with the osteopontins at 86%. The middle portion of the peptide had greatly reduced homology, ranging from 50% (amino acids 83-174) to 12% (amino acids 175-219). There were also deletions and additions of sequence in osteopontin-k that were not found in the other osteopontins. The homologies suggest that these proteins are highly related and may be derived from a common gene by alternative splicing. A 678 bp cRNA probe constructed from pBk2.1, containing a region with low homology to the osteopontins (amino acids 183-219 with less than 20% homology, plus amino acids 220-261 and untranslated sequence), was used in northern blots and RNAse protection assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Differential palmitoylation directs the AMPA receptor-binding protein ABP to spines or to intracellular clusters.

Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Construction of ovine adenovirus recombinants by gene insertion or deletion of related terminal region sequences.

An ovine adenovirus which may be the prototype for a new group of adenoviruses has been engineered as a gene transfer vector. One recombinant containing a 0.95-kb insertion expressed a sheep parasite antigen from the ovine adenovirus major late promoter and tripartite leader sequences. It was shown that insertions of at least 4.3 kb were tolerated at either one of two sites in the genome without the introduction of a compensating deletion. The unique structure of this viral genome was further emphasized by the discovery that four open reading frames at the right hand end show significant identity to each other but not to other sequences in the databases. Two other unrelated open reading frames were also present. RT-PCR analysis identified two transcripts in this region which were derived from a promoter which was located very close to, or within the ITR sequence. Splicing removed all but the first and last of the ORFs from these RNAs, suggesting that some sequences might be nonessential for replication in vitro. A approximately 2-kb deletion, which removed or truncated the internal reading frames was introduced into the region without affecting virus viability. The carrying capacity of OAV recombinants should therefore be at least 6.3 kb. The relative packaging capacity of OAV (114%) therefore exceeds that of Ad5 (105%), although a comparison of virus particle sizes by electron microscopy showed that OAV was smaller than Ad5. These studies improve the potential utility of OAV as a gene transfer vector.

Adenoviridae↗
PubMed1992

Structure of the human pancreatic cholesterol esterase gene.

The gene for human pancreatic cholesterol esterase consists of 11 exons and 10 introns and is 9.2 kb in length. The last and longest exon (841 nucleotides) is unique to the human gene. Functional amino acids are encoded on separate exons. The leader sequence is encoded by a single exon which carries two additional N-terminal amino acids of the mature functional protein. A positive TATA element is identified 43 nucleotides from the start codon. Pulse-field gel electrophoresis and hybridization with various cDNA probes and direct sequence data revealed the existence of a CEase-like gene. Partial sequence analysis of this gene from a human cosmid library and human genomic DNA showed a premature stop signal in exon 10, shortly after the codon for the active-site histidine. Both the functional gene and the CEase-like gene have a polyadenylation signal in the 3'-untranslated region. Thus, the complex gene structure for this intestinally active enzyme may provide in part a potential molecular explanation for the well-known heterogeneity of the intestinal absorption of cholesterol.

Base Sequence↗
PubMed1994

Sequence comparison between the flavoprotein subunit of the fumarate reductase (complex II) of the anaerobic parasitic nematode, Ascaris suum and the succinate dehydrogenase of the aerobic, free-living nematode, Caenorhabditis elegans.

Complex II in adult mitochondria of the parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron-transport observed in these organelles. In the present study, cDNAs for the flavoprotein (Fp) subunits of complex II have been isolated, cloned and sequenced from both A. suum and the aerobic, free-living nematode, Caenorhabditis elegans. Additional sequence at the 3' end of the mRNAs was determined by the Rapid Amplification of cDNA Ends (RACE). Nucleotide sequence analysis of the A. suum cDNAs revealed a 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1935 nucleotides and a 3' untranslated region of 616 nucleotides including a poly (A) tail from a polyadenylation signal (AATAAA). The open reading frame encoded a 645 amino acid sequence, including a 30 amino acid mitochondrial presequence. The amino acid sequences for the Fp subunits from both organisms were very similar, even though the ascarid enzyme functions physiologically as a fumarate reductase and the C. elegans enzyme a succinate dehydrogenase. The ascarid sequence was much less similar to the Escherichia coli fumarate reductase. The sensitivity of other Fp subunits to sulfhydryl reagents appears to reside in a cysteine immediately preceding a conserved arginine in the putative active site. In both nematode sequences, this cysteine is replaced by serine even though the succinate dehydrogenase activity of both enzymes is still sensitive to sulfhydryl inhibition. A cysteine six residues upstream of the serine may be involved in the sulfhydryl sensitivity of the nematode enzymes. Surprisingly, in contrast to succinate dehydrogenase activity, the fumarate reductase activity of the ascarid enzyme was not sensitive to sulfhydryl inhibition, suggesting that the mechanism of the two reactions involves separate catalytic processes.

Amino Acid Sequence↗
PubMed1993

The SpEGF III gene encodes a member of the fibropellins: EGF repeat-containing proteins that form the apical lamina of the sea urchin embryo.

We have identified a gene in the sea urchin Strongylocentrotus purpuratus that encodes a protein with multiple epidermal growth factor(EGF)-like motifs. The SpEGF III cDNA sequence predicts a 570 amino acid protein with a complex domain structure similar to that of the fibropellins--the protein products of the SpEGF I gene. A putative hydrophobic leader sequence is followed by an EGF-like motif, a domain similar to complement Cls, seven tandem EGF-like repeats, and a carboxy terminal domain similar to avidin. The 2.9-kb SpEGF III mRNA is expressed from a single copy gene. SpEGF III mRNA is present at low levels in unfertilized eggs and during early cleavage, then rapidly increases in abundance between late morula and mesenchyme blastula stages to maximal levels maintained through subsequent stages. Polyclonal antisera from SpEGF III fusion proteins reveal that the protein is a 100-kDa fibropellin which is present in unfertilized eggs but does not accumulate substantially until the mesenchyme blastula stage. The protein is localized to the apical lamina, a structurally complex component of the extracellular matrix that is made up of three major proteins, two of which are the differentially spliced products of SpEGF I. The size and localization of the SpEGF III protein, and the results of immunoprecipitation assays which reveal that it is tightly associated with the products of SpEGF I, indicate that it is the third major protein component of the apical lamina. The timing of SpEGF III protein accumulation is coincident with an increase in the structural complexity of the apical lamina, and with the developmental period when the apical lamina plays an important role in gastrulation.

Amino Acid Sequence↗
PubMed1997

Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo.

At least two hairpins in the 5' untranslated leader region, stem-loops 1 and 3 (SL1 and SL3), contribute to human immunodeficiency virus type 1 RNA encapsidation in vivo. We used a competitive assay, which measures the relative encapsidation efficiency of mutant viral RNA in the presence of competing wild-type RNA, to compare the contributions of SL1, SL3, and two adjacent secondary structures, SL2 and SL4, to encapsidation. SL2 is not required for RNA encapsidation, while SL1, SL3, and SL4 all contribute approximately equally to encapsidation. To determine whether these hairpins function in a position-dependent manner, we interchanged the positions of two of these stem-loop structures. This resulted in substantial diminution of encapsidation, indicating that the secondary structures that comprise E, the encapsidation signal, function only in their correct contexts. Mutation of nucleotides flanking SL1 and SL3 had little effect on encapsidation. We also showed that SL1, while present on both genomic and subgenomic viral RNAs, nonetheless contributes to selective encapsidation of genomic RNA. Taken together, these data are consistent with the formation of a higher-order RNA structure, partially composed of SL1, SL3, and SL4, that functions to effect concurrent encapsidation of full-length RNA and exclusion of subgenomic RNA. Finally, it has been reported that E is required for efficient translation of Gag mRNA in vivo. However, we have found that a variety of mutants, including a mutant lacking the entire region encompassing SL1, SL2, and SL3, still produce RNAs that are efficiently translated. These data indicate that E is unlikely to contribute to efficient Gag mRNA translation in vivo.

Base Sequence↗