Showing papers on "Intron published in 1991"

Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mrna splicing

Abstract: Rous sarcoma virus was shown to induce in chicken embryo fibroblasts (CEF) a 4.1-kilobase mRNA (designated CEF-147) encoding a 603-amino acid protein. Analysis of the protein sequence showed that it shared 59% amino acid identity with sheep prostaglandin G/H synthase, the enzyme that catalyzes the rate-limiting steps in the production of prostaglandins. Significant differences, at both the protein and mRNA levels, existed between the src oncogene product-inducible prostaglandin synthase and the protein isolated and cloned from sheep seminal vesicle, suggesting that the src-inducible prostaglandin synthase may be a new form of the enzyme. A distinguishing feature of src-inducible prostaglandin synthase mRNA is its low abundance in nonproliferating chicken embryo fibroblasts and its relatively high abundance in src-transformed cells. Additionally, the majority of the src-inducible prostaglandin synthase RNA present in nonproliferating cells was found to be nonfunctional because of the presence of an unspliced intron that separated the signal peptide from the remainder of the protein. Upon mitogenic stimulation, this intron was removed, resulting in the induction of fully-spliced CEF-147 mRNA.

The Vascular Endothelial Growth Factor Family: Identification of a Fourth Molecular Species and Characterization of Alternative Splicing of RNA

Abstract: Vascular endothelial growth factor (VEGF) was recently identified as a secreted, direct-acting mitogen specific for vascular endothelial cells and capable of stimulating angiogenesis in vivo. Molecular cloning revealed multiple forms of VEGF, apparently arising from alternative splicing of its RNA transcript. We have examined various human cDNA libraries by the polymerase chain reaction technique and discovered a fourth molecular form, VEGF206. This form contains a 41-amino acid insertion relative to the most abundant form, VEGF165, and includes the highly basic 24-amino acid insertion found in VEGF189. Southern blot analysis revealed that a single gene encoded these various forms, and nucleic acid sequence analysis of a portion of the VEGF gene revealed an intron/exon structure compatible with alternative splicing of RNA as a mechanism for their generation. Transient transfection of human embryonic kidney 293 cells showed that, like VEGF189, VEGF206 was predominately cell-associated and only very poorly secreted despite the presence of the signal peptide identical to that found in VEGF121 and VEGF165, both of which are efficiently exported from the cell. Vascular permeability activity was detected in the medium of 293 cells transfected with all four forms of VEGF; however, endothelial cell mitogenic activity was apparent only with VEGF121 and VEGF165. Thus, alternative splicing of VEGF RNA can produce four polypeptides with strikingly different secretion patterns, which suggests multiple physiological roles for this family of proteins.

Biochemical Mechanisms of Constitutive and Regulated Pre-mRNA Splicing

Abstract: INTRODUCTION 560 SPLICING PATHWAy 560 THE PARADIGM: AUTOCATALYTIC SPLICING OF GROUP II INTRONS 560 SPLICEOSOME ASSEMBLY PATHWAY 561 The Vi snRNP-Binding Reaction ...... . ... . 563 The U2 snRNP-Binding Reaction 563 Entry of U4/U5/U6 snRNPs 565 Destabilization of V4 snRNA 565 THE SPLICING PROCESS 566 First Step and Catalysis 566 Second Step 566 mRNA Release, Spliceosome Disassembly, and Intron Degradation 567 U snRNPs 568 Sm-Binding Site , . . . . . . . . . . . . 568 Cap Structure , . . . . . . . . . . . . 569 snRN P Assembly 569 Structure-Function Analysis of Spliceosomal sRNAs 569 Higher Order snRNP-snRNP Interactions 574 NON-snRNP SPLICING FACTORS 574 Mammalian Factors , . . . . . . . . . . . . 574 Yeast Factors .. .... .. . .. .. 578 Protein Motifs in Splicing Factors 579 SELECTION OF SPLICE SITES 580 Splice Site Selection: A Hierarchy of Competition 581 Other Models 581

Polymorphic structure of the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the first intron of the human TNF-beta gene correlates with a variant amino acid in position 26 and a reduced level of TNF-beta production.

Abstract: Since a dysregulated synthesis of tumor necrosis factor alpha (TNF-alpha) may be involved in the pathogenesis of autoimmune diseases, it was of interest to precisely locate the recently reported NcoI restriction fragment length polymorphism (RFLP) of the TNF-alpha region. However, by mapping of 56.8 kb of overlapping cosmid clones and direct sequencing, we could localize the polymorphic NcoI restriction site within the first intron of the TNF-beta gene and not in the TNF-alpha gene. To study whether regulatory mechanisms are affected by this polymorphism, we analyzed the TNF-alpha/TNF-beta production of phytohemagglutinin-stimulated peripheral blood mononuclear cells of individuals homozygous for the TNF-beta NcoI RFLP by ELISA and concomitant Northern blot analysis. On days 2-4 after stimulation with mitogen, the TNFB*1 allele corresponding to a 5.3-kb NcoI fragment presented with a significantly higher TNF-beta response. A mRNA analysis demonstrated that higher protein levels of TNF-beta correlate also with increased amounts of TNF-beta transcripts. No allelic association was found in respect to TNF-alpha production. To further investigate a possible allelic influence on transcription, we determined the DNA sequence of 2 kb of the 5' portion of our cloned TNFB*2 allele and compared it with the available TNF-beta sequences. By computer-aided recognition motif search of DNA binding factors, we report putative binding sites conserved between mouse and man in the 5' flanking region as well as in intron 1 of the TNF-beta gene, found also in other cytokine promoter sequences. In addition, by polymerase chain reaction amplification and sequencing of 740 bp of the 5' part of TNF-beta of individuals typed homozygously for the NcoI RFLP, we could show that amino acid position 26 is conserved as asparagine in the TNFB*1 and as threonine in the TNFB*2 sequence. A previously reported, EcoRI RFLP in the 3' untranslated region of TNF-beta does not segregate with either of the two alleles. Thus, four TNFB alleles can be defined at the DNA level.

Heterologous introns can enhance expression of transgenes in mice.

Abstract: In a previous study we showed that genomic constructs were expressed more efficiently in transgenic mice than constructs that were identical except for the lack of introns. Using the mouse metallothionein promoter-rat growth hormone gene construct as a model, we show that the first intron of the rat growth hormone gene is essential for high-level expression, whereas the other three introns are less effective. Several heterologous introns placed 3' of the coding region of an intronless rat growth hormone gene are also ineffective. However, insertion of some heterologous introns between the metallothionein promoter and the growth hormone gene improves expression. To determine whether addition of heterologous introns would provide a general strategy for improving expression, we have tested them in conjunction with other intronless genes and with different promoters.

CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location.

Abstract: CD28, initially detected on human T lymphocytes with the help of antibodies, and CTLA-4, obtained by reverse genetics through its preferential expression in mouse activated T cells, are both single-V domain members of the Ig superfamily. Early work showed a relationship between these two molecules, which we wished to further document, in particular because of the growing realization of the functional importance of CD28 in some T cell activation pathways. Isolation and analysis of the mouse CTLA-4 gene and further analysis of the human CTLA-4 gene showed that both of these and the human CD28 gene share the same overall intron/exon organization. The nucleic acid sequence homology of the exons was found to extend across both molecules and species, whereas the 5' and 3' flanking regions exhibited homology across species but not between molecules. Message expression of human CTLA-4 was only detected in activated T cells and, thus, shares with that of mouse CTLA-4 and of mouse and human CD28 a lymphoid tissue distribution, although apparently broader for the latter. Two main human CTLA-4 transcripts of about 1.8 and 0.8 kb were detected, the smaller of which may derive, as reported for human CD28, from the use of an alternate degenerated polyadenylation signal sequence. The nucleic acid sequence data allowed a direct comparison of the four putative complete protein sequences of CD28 and CTLA-4 in the mouse and the human, showing striking homologies, especially in some stretches (such as a MYPPPY hexamer in the hinge region) conserved across molecules and across species. The mouse CD28 gene was localized to chromosome 1 band C by in situ hybridization with three different radioactive probes, indicating, together with previous data, that the CD28 and CTLA-4 genes map to the same chromosomal region in both the mouse and the human. Thus, CD28 and CTLA-4 were found to be strikingly similar in most respects, in terms of structure, sequence, expression, and gene location, furthermore in two species, strongly suggesting that their genes are the direct products of a duplication event and raising the possibility of functional homologies between the corresponding proteins.

Exon amplification: a strategy to isolate mammalian genes based on RNA splicing.

Abstract: We have developed a method, exon amplification, for fast and efficient isolation of coding sequences from complex mammalian genomic DNA. This method is based on the selection of RNA sequences, exons, which are flanked by functional 5' and 3' splice sites. Fragments of cloned genomic DNA are inserted into an intron, which is flanked by 5' and 3' splice sites of the human immunodeficiency virus 1 tat gene contained within the plasmid pSPL1. COS-7 cells are transfected with these constructs, and the resulting RNA transcripts are processed in vivo. Splice sites of exons contained within the inserted genomic fragment are paired with splice sites of the flanking tat intron. The resulting mature RNA contains the previously unidentified exons, which can then be amplified via RNA-based PCR and cloned. Using this method, we have isolated exon sequences from cloned genomic fragments of the murine Na,K-ATPase alpha 1-subunit gene. We have also screened randomly selected genomic clones known to be derived from a segment of human chromosome 19 and have isolated exon sequences of the DNA repair gene ERCC1. The sensitivity and ease of the exon amplification method permit screening of 20-40 kilobase pairs of genomic DNA in a single transfection. This approach will be extremely useful for rapid identification of mammalian exons and the genes from which they are derived as well as for the generation of chromosomal transcription maps.

The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain.

Abstract: To determine the mechanisms by which multiple forms of fibroblast growth factor (FGF) receptors are generated, we have mapped the arrangement of exons and introns in the human FGF receptor 1 (FGFR 1) gene (flg). We found three alternative exons encoding a portion of the third immunoglobulin (Ig)-like domain of the receptor. One of these alternatives encodes a sequence that is part of a secreted form of FGFR 1. The other two encode sequences that are likely part of transmembrane forms of FGFR 1. One of these forms has not been previously reported in published cDNAs. Also, we have determined the structural organization of a portion of the human FGFR 2 gene (bek) and found a similar arrangement of alternative exons for the third Ig-like domain. The arrangement of these genes suggests that there are conserved mechanisms governing the expression of secreted FGF receptors as well as the expression of at least two distinct membrane-spanning forms of the FGF receptors. The diverse forms appear to be generated by alternative splicing of mRNA and selective use of polyadenylation signals.

Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells

Abstract: A 2.4 kb fragment of hCMV (Towne strain), containing the 5' end of the major immediate-early gene, has been cloned, sequenced, and used to construct a series of mammalian cell expression plasmids. The effects of regulatory regions present on this fragment were assessed using human glycoproteins as reporter molecules. We compared secreted levels of Factor VIII, t-PA, and HIV-1 envelope glycoproteins in cells transfected with plasmids in which intron A of the immediate-early gene was present or absent. Secretion of several glycoproteins was significantly higher when cells were transfected with intron A-containing plasmids. Mutation of three basepairs in the strong nuclear factor 1 (NF1) binding site in intron A led to reduced transient expression levels, but not to the level observed in the absence of intron A. Reduced expression from NF1 mutant plasmids was roughly correlated with reduced binding in vitro of NF1 proteins to a synthetic oligonucleotide containing the mutation. The evidence indicates that sequences in intron A positively regulate expression from the hCMV immediate-early enhancer/promoter in transformed monkey kidney cells.

A generic intron increases gene expression in transgenic mice.

Abstract: To investigate the role of splicing in the regulation of gene expression, we have generated transgenic mice carrying the human histone H4 promoter linked to the bacterial gene for chloramphenicol acetyltransferase (CAT), with or without a heterologous intron in the transcription unit. We found that CAT activity is 5- to 300-fold higher when the transgene incorporates a hybrid intron than with an analogous transgene precisely deleted for the intervening sequences. This hybrid intron, consisting of an adenovirus splice donor and an immunoglobulin G splice acceptor, stimulated expression in a broad range of tissues in the animal. Although the presence of the hybrid intron increased the frequency of transgenics with significant CAT activity, it did not affect the integration site-dependent variation commonly seen in transgene expression. To determine whether the enhancement is a general outcome of splicing or is dependent on the particular intron, we also produced equivalent transgenics carrying the widely used simian virus 40 small-t intron. We found that the hybrid intron is significantly more effective in elevating transgene expression. Our results suggest that inclusion of the generic intron in cDNA constructs may be valuable in achieving high levels of expression in transgenic mice.

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein.

Abstract: The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.

Visualizing the higher order folding of a catalytic RNA molecule.

Abstract: The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.

Complete Structure of the Human Gene for 92-kDa Type IV Collagenase

Abstract: The complete structure of the human gene for 92kDa type IV collagenase was determined. Two overlapping genomic clones spanning 26 kilobases (kb) of genomic DNA were shown to contain the entire 7.7-kb structural gene together with 15 and 3.5 kb of 5’-end and 3’-end flanking regions, respectively. The 92-kDa type IV collagenase gene contains 13 exons as does the 72-kDa type IV collagenase gene. All intron locations of the 92-kDa enzyme gene coincided with intron locations in the 72-kDa enzyme gene. Exons 5,6, and 7 which were 174, 174, and 177 base pairs long, respectively, each encoded one complete internal repeat which resembles the collagen-binding domains of fibronectin. The sequence coding for a unique 48-residue segment in the 92-kDa type IV collagenase that has no counterpart in other metalloproteinases was not present in a separate exon, but was contained in exon 9 which also codes for sequences with homology to the

Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes

Abstract: The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome. The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing. The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases. A putative RNA-binding motif originally identified in bacterial ribosomal protein SI and Escherichia coli polynucleotide phosphorylase has also been found in PRP22.

Tomato spotted wilt virus L RNA encodes a putative RNA polymerase.

Abstract: The complete nucleotide sequence of the large (L) genome segment of tomato spotted wilt virus (TSWV) has been determined. The RNA is 8897 nucleotides long and contains complementary 3' and 5' ends, comprising 62 nucleotides at the 5' end and 66 nucleotides at the 3' end. The RNA is of negative polarity, with one large open reading frame (ORF) located on the viral complementary strand. This ORF corresponds to a primary translation product of 2875 amino acids in length, with a predicted Mr of 331,500. Comparison with the polymerase proteins of other negative-strand viruses indicates that this protein most likely represents the viral polymerase. The genetic organization of TSWV L RNA is similar to that of the L RNA segments of Bunyamwera and Hantaan viruses, animal-infecting representatives of the Bunyaviridae.

Five easy pieces

Abstract: Why should wait for some days to get or receive the five easy pieces book that you order? Why should you take it if you can get the faster one? You can find the same book that you order right here. This is it the book that you can receive directly after purchasing. This five easy pieces is well known book in the world, of course many people will try to own it. Why don't you become the first? Still confused with the way?

Single-step selection for Ty1 element retrotransposition

Abstract: The yeast retrotransposon Ty1 has been tagged with a reporter gene that allows selection of RNA-mediated transposition events and is applicable to the study of retroelements in other organisms. The reporter gene is a yeast HIS3 gene interrupted by an artificial intron (AI) in the antisense orientation. The HIS3AI sequences were inserted into a Ty1 element such that the intron is on the sense strand of the Ty1 element; therefore, splicing and retrotransposition of marked Ty1 transcripts can give rise to His+ cells. Fusion of the Ty1-H3mHIS3AI element to the inducible GAL1 promoter resulted in a high frequency of histidine prototrophs upon galactose induction. Moreover, spontaneous His+ revertants derived from strains containing genomic TymHIS3AI elements are a result of retrotransposition. By using this assay, we estimated the Ty1 transposition rate to be between 3 x 10(-7) and 1 x 10(-5) transpositions per Ty1 element per generation. Variations in the transposition rate of individual Ty1 elements are correlated with the relative abundance of their transcripts.

Antibiotic inhibition of group I ribozyme function.

Abstract: THE discovery of catalytically active RNA has provided the basis for the evolutionary concept of an RNA world. It has been proposed that during evolution the functions of ancient catalytic RNA were modulated by low molecular weight effectors, related to antibiotics, present in the primordial soup. Antibiotics and RNA may have coevolved in the formation of the modern ribosome1. Here we report that a set of aminoglycoside antibiotics, which are known to interact with the decoding region of the 16S ribosomal RNA of Escherichia coli2–4, inhibit the second step of splicing of the T4 phage-derived td intron. Thus catalytic RNA seems to interact not only with a mononucleotide5 and an amino acid6, but also with another class of biomolecules, the sugars. Splicing of other group I introns but not group II introns was inhibited. The similarity in affinity and specificity of these antibiotics for group I introns and rRNAs may result from recognition of evolutionarily conserved structures.

Alternatively spliced products of the maize P gene encode proteins with homology to the DNA-binding domain of myb-like transcription factors

Abstract: The Zea mays P gene has been postulated to regulate the biosynthetic pathway of a flavonoid-derived pigment in certain floral tissues [Styles, E D & Ceska, O (1977) Can J Genet Cytol 19, 289-302] We have characterized two P transcripts that are alternatively spliced at their 3' ends One message of 1802 nucleotides encodes a 437-kDa protein with an N-terminal region showing approximately 40% homology to the DNA-binding domain of several members of the myb family of protooncogene proteins A second message of 945 nucleotides encodes a 173-kDa protein that contains most of the myb-homologous domain but differs from the first protein at the C terminus The deduced P-encoded proteins show an even higher homology (70%) in the myb-homologous domain to the maize regulatory gene C1 Additionally, the P and C1 genes are structurally similar in the sizes and positions of the first and second exons and first intron We show that P is required for accumulation in the pericarp of transcripts of two genes (A1 and C2) encoding enzymes for flavonoid biosynthesis--genes also regulated by C1 in the aleurone

Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors.

Abstract: We have used in situ hybridization and immunocytochemistry to compare the nuclear localization of a specific nascent pre-mRNA and the essential non-snRNP splicing factor SC-35 Nascent c-fos transcripts were detected in serum-induced mouse fibroblasts by in situ hybridization with genomic c-fos probes Prior to serum induction no c-fos RNA is detected, but these transcripts localize to two dots in the interphase nucleus after induction The time course of appearance of the dots correlates with the previously determined time course of transcriptional activation of the gene Upon further analysis by confocal laser scanning microscopy, we have determined that the dots extend through the depth of the nucleus, forming paths By using high-voltage electron microscopy, we have found that the c-fos path extends out and comes into direct contact with the nuclear envelope We have also compared the localization of c-fos transcripts with the speckled nuclear regions that are enriched in snRNPs and the non-snRNP splicing factor SC-35 Direct observations of three-dimensional rotations have revealed a close association between the c-fos transcripts and the nuclear speckles This study demonstrates a direct link between specific nascent RNA transcripts and nuclear speckles that are enriched in pre-mRNA splicing factors

Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants

Abstract: We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast. Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required. Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential. Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia. As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize. However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal. The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots. We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts. The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron.

Human von Willebrand factor gene and pseudogene: structural analysis and differentiation by polymerase chain reaction

Abstract: Structural analysis of the von Willebrand factor gene located on chromosome 12 is complicated by the presence of a partial unprocessed pseudogene on chromosome 22q11-13. The structures of the von Willebrand factor pseudogene and corresponding segment of the gene were determined, and methods were developed for the rapid differentiation of von Willebrand factor gene and pseudogene sequences. The pseudogene is 21-29 kilobases in length and corresponds to 12 exons (exons 23-34) of the von Willebrand factor gene. Approximately 21 kilobases of the gene and pseudogene were sequenced, including the 5{prime} boundary of the pseudogene. The 3{prime} boundary of the pseudogene lies within an 8-kb region corresponding to intron 34 of the gene. The presence of splice site and nonsense mutations suggests that the pseudogene cannot yield functional transcripts. The pseudogene has diverged {approximately}3.1{percent} in nucleotide sequence from the gene. This suggests a recent evolutionary origin {approximately}19-29 million years ago, near the time of divergence of humans and apes from monkeys. Several repetitive sequences were identified, including 4 Alu, one Line-1, and several short simple sequence repeats. Several of these simple repeats differ in length between the gene and pseudogene and provide useful markers for distinguishing these loci. Sequence differences betweenmore » the gene and pseudogene were exploited to design oligonucleotide primers for use in the polymerase chain reaction to selectivity amplify sequences corresponding to exons 23-34 from either the von Willebrand factor gene or the pseudogene. This method is useful for the analysis of gene defects in patients with von Willebrand disease, without interference from homologous sequences in the pseudogene.« less