Showing papers on "Intron published in 1984"
TL;DR: In this paper, a simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described, based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter.
Abstract: A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).
5,732 citations
TL;DR: The complete 186,000 base-pair (bp) human factor VIII gene has been isolated and consists of 26 exons ranging in size from 69 to 3,106 bp and introns as large as 32.4 kilobases as mentioned in this paper.
Abstract: The complete 186,000 base-pair (bp) human factor VIII gene has been isolated and consists of 26 exons ranging in size from 69 to 3,106 bp and introns as large as 32.4 kilobases (kb). Nine kb of mRNA and protein-coding DNA has been sequenced and the mRNA termini have been mapped. The relationship between internal duplications in factor VIII and evolution of the gene is discussed.
988 citations
TL;DR: Results imply that sequences within IVS1 actively participate in splicing, and it is proposed that the 5' end ofIVS1 is joined by a 2'-5' phosphodiester linkage to the A residue in the RNAase T1 oligonucleotide ACTCTCTCTG located 28-37 nucleotides upstream from the IVS 1 3' end.
618 citations
TL;DR: Nucleotide sequence analysis in conjunction with primer extension and S1 nuclease protection experiments show that the structure of the rp49 gene consists of a 102 bp 5' exon, a single 59 bp intron, and a 420 bp 3'Exon, encoding a total of 132 amino acids.
Abstract: In this communication, we describe several features of the D. melanogaster gene which codes for ribosomal protein 49 (rp49). Nucleotide sequence analysis in conjunction with primer extension and S1 nuclease protection experiments show that the structure of the rp49 gene consists of a 102 bp 5' exon, a single 59 bp intron, and a 420 bp 3' exon, encoding a total of 132 amino acids. The rp49 gene shares many features with other abundantly expressed Drosophila genes, including codon preference, which are discussed.
495 citations
TL;DR: Interestingly, those portions of the polypeptide chain predicted to form loops on the cytoplasmic face of rhodopsin are perfectly conserved between the human and bovine proteins.
Abstract: We have isolated and completely sequenced the gene encoding human rhodopsin. The coding region of the human rhodopsin gene is interrupted by four introns, which are located at positions analogous to those found in the previously characterized bovine rhodopsin gene. The amino acid sequence of human rhodopsin, deduced from the nucleotide sequence of its gene, is 348 residues long and is 93.4% homologous to that of bovine rhodopsin. Interestingly, those portions of the polypeptide chain predicted to form loops on the cytoplasmic face of rhodopsin are perfectly conserved between the human and bovine proteins.
475 citations
TL;DR: The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5' end of the intervening sequence joined to a site near the 3' splice site, which has been characterized for an adenovirus 2 major late transcript.
Abstract: The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5' end of the intervening sequence joined to a site near the 3' splice site. This lariat structure, which has been characterized for an adenovirus 2 major late transcript, has a branch point, with 2'-5' and 3'-5' phosphodiester bonds emanating from a single adenosine residue. The excised intervening sequence retains the branch site and terminates in a guanosine residue with a 3' hydroxyl group. The phosphate group at the splice junction between the two exons originates from the 3' splice site at the precursor.
404 citations
TL;DR: The position and size of introns, the overall base composition, and the codon preference for the alpha 1-anti-trypsin gene differ from those for the chicken ovalbumin gene even though the two proteins belong to a common protein family, as judged by amino acid sequence homology.
Abstract: A 1434 base pair human liver cDNA coding for the entire alpha 1-antitrypsin protein has been isolated and sequenced. Translation of the coding region into amino acids reveals a precursor molecule which contains a 24 amino acid signal peptide and 394 amino acids present in the mature polypeptide chain. The human gene for the S variant of alpha 1-antitrypsin has also been subcloned and sequenced. The gene is composed of 10226 nucleotide bases and is approximately equimolar for all 4 nucleotides. The gene contains four intervening sequences (introns) and 5' and 3' noncoding regions which are 54 and 79 nucleotides in length, respectively. A 5.3-kilobase intron exists in the 5' noncoding region and contains a 143 amino acid open reading frame, an Alu family sequence, and a pseudo transcription initiation region. No significant differences in base composition are seen between the introns and those regions corresponding to coding regions of the corresponding mRNA (exons). A sequence of 1951 nucleotides flanking the 5' end of the gene has also been determined and contains a "TATA" box sequence (TTAAA-TA) 21 nucleotides upstream from the proposed transcription start site. Comparison of the gene sequence with the cDNA sequence reveals a single base substitution (A----T), which results in a Glu----Val substitution at position 264 in the S variant protein. The position and size of introns, the overall base composition, and the codon preference for the alpha 1-anti-trypsin gene differ from those for the chicken ovalbumin gene even though the two proteins belong to a common protein family, as judged by amino acid sequence homology.
367 citations
TL;DR: Mouse MT-I and MT-II mRNAs are induced to approximately the same extent in vivo in response to cadmium, dexamethasone, or lipopolysaccharide and are regulated by metals but not by glucocorticoids after transfection into HeLa cells.
Abstract: The mouse metallothionein II (MT-II) gene is located approximately 6 kilobases upstream of the MT-I gene. A comparison of the sequences of mouse MT-I and MT-II genes (as well as those of other mammals) reveals that the coding regions are highly conserved even at "silent" positions but that the noncoding regions and introns are extremely divergent between primates and rodents. There are four blocks of conserved sequences in the promoters of mouse MT-I, mouse MT-II, and human MT-IIA genes; one includes the TATAAA sequence, and another has been implicated in regulation by heavy metals. Mouse MT-I and MT-II mRNAs are induced to approximately the same extent in vivo in response to cadmium, dexamethasone, or lipopolysaccharide. Mouse MT-I and MT-II genes are regulated by metals but not by glucocorticoids after transfection into HeLa cells.
343 citations
TL;DR: Direct evidence is shown for a functionally similar enhancer within the large κ gene intron of the mouse which is, however, less active than the heavy-chain gene enhancer.
Abstract: During differentiation of lymphocytes into antibody-producing cells, an immunoglobulin kappa variable-region gene is transcriptionally activated by rearrangement linking it to a kappa constant (C kappa) region gene which is already transcribed prior to somatic rearrangement. The presence of a transcriptional enhancer element within the large intron of the kappa light-chain gene has been postulated to explain this mode of activation, supported by evidence of a chromatin region which is preferentially accessible to DNase I and restriction enzymes. This DNA region contains a segment of about 130 base pairs (bp) which is strongly conserved between mouse, rabbit and man. Moreover, no transcripts are detectable from a kappa gene, which is truncated within the large intron. Recently, a lymphocyte-specific enhancer has been identified downstream of the joining region in immunoglobulin heavy-chain genes. We now show direct evidence for a functionally similar enhancer within the large kappa gene intron of the mouse. It is, however, less active than the heavy-chain gene enhancer. In contrast, no enhancer was found to be associated with a cloned lambda I light-chain gene.
329 citations
TL;DR: The mRNA sequence of the human intrinsic clotting factor IX (Christmas factor) has been completed and is 2802 residues long, including a 29 residue long 5′ non‐coding and a 1390 residue long 3′ non-coding region, but excluding the poly(A) tail.
Abstract: The mRNA sequence of the human intrinsic clotting factor IX (Christmas factor) has been completed and is 2802 residues long, including a 29 residue long 5' non-coding and a 1390 residue long 3' non-coding region, but excluding the poly(A) tail. The factor IX gene is approximately 34 kb long and we define, by the sequencing of 5280 residues, the presumed promoter region, all eight exons, and some intron and flanking sequence. Introns account for 92% of the gene length and the longest is estimated to be 10 100 residues. Exons conform roughly to previously designated protein regions, but the catalytic region of the protein is coded by two separate exons. This differs from the arrangement in the other characterized serine protease genes which are further subdivided in this region.
324 citations
TL;DR: A genomic clone carrying the human tissue-type plasminogen activator (t-PA) gene was isolated from a cosmid library, and the gene structure was elucidated by restriction mapping, Southern blotting, and DNA sequencing, revealing a gene organization similar to other serine proteases.
Abstract: A genomic clone carrying the human tissue-type plasminogen activator (t-PA) gene was isolated from a cosmid library, and the gene structure was elucidated by restriction mapping, Southern blotting, and DNA sequencing. The cosmid contained all the coding parts of the mRNA, except for the first 58 bases in the 5' end of the mRNA, and had a total length of greater than 20 kilobases. It was separated into at least 14 exons by at least 13 introns, and the exons seemed to code for structural or functional domains. Thus, the signal peptide, the propeptide, and the domains of the heavy chain, including the regions homologous to growth factors, and to the "finger" structure of fibronectin, are all encoded by separate exons. In addition, the two kringle regions of t-PA were both coded for by two exons and were cleaved by introns at identical positions. The region coding for the light chain, comprising the serine protease part of the molecule was split by four introns, revealing a gene organization similar to other serine proteases.
TL;DR: The most abundant Epstein-Barr virus mRNA in a latently infected cell line, IB4, was a 2,8-kilobase RNA encoded by largely unique DNA near the right end of the genome, which could be responsible for the new antigen detected in the plasma membrane of Epstein- Barr virus-transformed cells, lymphocyte-determined membrane antigen.
Abstract: The most abundant Epstein-Barr virus mRNA in a latently infected cell line, IB4, established by in vitro growth transformation with virus, was a 2,8-kilobase RNA encoded by largely unique DNA near the right end of the genome. The RNA was transcribed from right to left, and two introns were spliced out. This region of the genome was sequenced, and the exons of the RNA were identified by S1 analysis of DNA-RNA hybrids and primer extension. The first start codon in the RNA was 40 nucleotides from its 5' end. Beginning with the start codon, there was a 1,158-nucleotide open reading frame which crossed both introns. The important characteristics of the translated protein were as follows. (i) The amino terminus was highly charged and not suggestive of a leader sequence. (ii) There were six markedly hydrophobic alpha-helical domains, each having 21 amino acids and connected by 5 to 7 amino acid segments predicted to be reverse turns. (iii) The carboxy-terminal 200 amino acids were markedly acidic, containing 6 glutamic and 37 aspartic acids. The hydrophobic region is predicted to form six membrane-spanning regions, leaving the short charged amino terminus and long acidic carboxy terminus on the inside of the membrane. This protein could be responsible for the new antigen detected in the plasma membrane of Epstein-Barr virus-transformed cells, lymphocyte-determined membrane antigen. There were two other open reading frames in the RNA.
TL;DR: It is noted that a single-stranded region of small nuclear RNA U2 contains sequences complementary both to the proposed mammalian internal signal and to the neighboring CT-A-G at the 3' intron boundary, and a role for U2 ribonucleoprotein in intron splicing is suggested.
Abstract: Splicing of introns of yeast pre-mRNAs requires an internal conserved sequence T-A-C-T-A-A-C that is located 20-55 nucleotides from the 3' intron boundary. Sequences differing only in certain positions from this yeast signal have now been identified in the corresponding internal region of pre-mRNA introns of a variety of animal genes. A computer program that searches for homologues to a consensus structure and calculates the accuracy of match of each homologue is used to locate these sequences. We list here the signals found by this search in introns of sea urchin, mouse, rat, and human genes and give the consensus for each species. We also give the consensus found for Drosophila and chicken and duck signals. We then discuss the accumulating evidence that these internal signals are required for splicing in animals. It is also noted that a single-stranded region of small nuclear RNA U2 contains sequences complementary both to the proposed mammalian internal signal and to the neighboring CT-A-G at the 3' intron boundary. A role for U2 ribonucleoprotein in intron splicing is thus suggested.
TL;DR: Four distinct forms of the excised actin intron are found in poly(A)- RNA from yeast carrying this transcription unit on a multicopy plasmid, and one of these contains a block to reverse transcription at the conserved UACUAAC sequence (TACTAAC box).
TL;DR: No specific internal intron sequences appear to be needed but a minimal intron length is important in rabbit beta-globin genes; extending the miniintron with polyoma or pBR322 fragments to 80 or more nucleotides restored normal splicing.
TL;DR: It is shown that the chemically synthesized decanucleotide 5'-TGTACTAACA-3', when introduced into a hybrid gene forming unspliceable RNA molecules, results in the generation of spliceable transcripts.
TL;DR: Analyses of the product with RNAase T1 revealed that pairing initiates at or near the 5' end of RNA I and propagates toward its 3' end, which facilitates complete pairing along the entire length ofRNA I that initiates from the 5'-end region.
TL;DR: The positions of these insertions and those of previously characterized insertions associated with six other mutations suggest that some insertions within an intron may still allow the production of correctly spliced RNA, but affect the amount, and correspondingly the expression of the w locus.
TL;DR: Comparison of Rev-A, Rev-T, and c-rel indicates that the v-rel sequences may have been transduced from the c-Rel (turkey) locus by a novel mechanism, and there are many differences between the amino acid sequences of the predicted translational products of v- Rel andc-rel which may account for their difference in transformation potential.
Abstract: Reticuloendotheliosis virus strain T (Rev-T) is a highly oncogenic replication-defective retrovirus which contains the oncogene v-rel. It is thought that Rev-T arose when a virus similar to Rev-A, the helper virus of Rev-T, infected a turkey and recombined with c-rel from that turkey. There is one large c-rel locus in the turkey genome which contains all of the sequences homologous to v-rel (K. C. Wilhelmsen and H. M. Temin, J. Virol. 49:521-529, 1984). We have sequenced v-rel and its flanking sequences, each of the regions of the c-rel locus from turkey that are homologous to v-rel and their flanking sequences, and the coding sequence for env and part of pol of Rev-A. The v-rel coding sequences can be translated into a 503-amino acid env-v-rel-out-of-frame-env fusion polypeptide. We have not detected any sequences in the Los Alamos or University of California-San Diego data bases that are more significantly related to the amino acid or nucleic acid sequence of v-rel than to the randomized sequence of v-rel. Comparison of Rev-A, Rev-T, and c-rel indicates that the v-rel sequences may have been transduced from the c-rel (turkey) locus by a novel mechanism. There are sequences in Rev-A and c-rel that are similar to splicing signals, indicating that the 5' virus-rel junction of Rev-T may have been formed by cellular RNA splicing machinery. Eight presumed introns have presumably been spliced out of c-rel to generate v-rel. There are also short imperfect regions of homology between sequences at the boundaries of v-rel and sequences in Rev-A and c-rel (turkey), indicating that c-rel may have been transduced by homologous recombination. There are many differences between the amino acid sequences of the predicted translational products of v-rel and c-rel which may account for their difference in transformation potential. These sequence differences between v-rel and c-rel include 10 missense transitions, four missense transversions, and three places where Rev-T has a small in-frame deletion of sequences relative to c-rel. Most of the coding sequence differences between c-rel and v-rel are nonconservative amino acid changes.
TL;DR: The complete nucleotide sequence of a 2.9-kb DNA fragment containing the CDC2 gene-complementing activity from Schizosaccharomyces pombe has been determined and results strongly suggest that the two genes code for similar functions.
TL;DR: Reassortment of functional modules of coding and regulatory sequence from preexisting viral or cellular sources, perhaps via RNA recombination, may be an important mechanism in RNA virus evolution.
Abstract: The plant viruses alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) each divide their genetic information among three RNAs while tobacco mosaic virus (TMV) contains a single genomic RNA. Amino acid sequence comparisons suggest that the single proteins encoded by AMV RNA 1 and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 are related to the NH2-terminal two-thirds and the COOH-terminal one-third, respectively, of the largest protein encoded by TMV. Separating these two domains in the TMV RNA sequence is an amber termination codon, whose partial suppression allows translation of the downstream domain. Many of the residues that the TMV read-through domain and the segmented plant viruses have in common are also conserved in a read-through domain found in the nonstructural polyprotein of the animal alphaviruses Sindbis and Middelburg. We suggest that, despite substantial differences in gene organization and expression, all of these viruses use related proteins for common functions in RNA replication. Reassortment of functional modules of coding and regulatory sequence from preexisting viral or cellular sources, perhaps via RNA recombination, may be an important mechanism in RNA virus evolution.
TL;DR: Human urokinase cDNA clones have been identified from a cDNA library prepared from total RNA of human fibroblasts transformed by simian virus 40 and the nucleotide sequence of one of the clones identifies this as a copy of a partially spliced polyadenylylated precursor to uro Kinase mRNA.
Abstract: Human urokinase cDNA clones have been identified from a cDNA library prepared from total RNA of human fibroblasts transformed by simian virus 40 [Okayama, H. & Berg, P. (1983) Mol. Cell. Biol. 3, 280-289]. Synthetic oligonucleotides, corresponding to urokinase protein sequence, were used as probes. The cloned cDNA covers most of the coding sequence and the entire 3' untranslated region. The nucleotide sequence of one of the clones identifies this as a copy of a partially spliced polyadenylylated precursor to urokinase mRNA. The introns separate functionally different domains of the enzyme. Human urokinase mRNA has been identified by RNA blot and its size was estimated at 2500 nucleotides.
TL;DR: DNA sequence analysis of these regions revealed that spectinomycin resistance results from a C/G to T/A transition at position 1192 of a 16S RNA gene, and alteration in 23S RNA identifies sequences important to peptidyl transfer.
Abstract: Recombinant DNA and classic genetic procedures were used to map a spectinomycin resistance mutation to a 121 base pair region of a 16S RNA gene and a macrolide-lincosamide-streptogramin type B resistance mutation to a 32 base pair region of a 23S RNA gene. DNA sequence analysis of these regions revealed that spectinomycin resistance results from a C/G to T/A transition at position 1192 of a 16S RNA gene. Resistance to macrolide, lincosamide and streptogramin type B antibiotics results from an A/T to T/A transversion at position 2058 of a 23S RNA gene. The alteration in 16S RNA is in a sequence that can participate in alternate base pairing arrangements that have been proposed to be involved in the translocation process. The alteration in 23S RNA identifies sequences important to peptidyl transfer.
TL;DR: Results suggest that the splicing of mRNA precursors may involve sites in the intervening sequence, cleavage at the 5' splice site, cleaving at the 3' splicing site, and ligation of the two exons.
TL;DR: Part of the sequence of which was captured in Moloney murine leukemia virus to generate the transforming gene (v-abl) of the Abelson murines leukemia virus has been isolated and characterized and indicates that v-abl derived from c-abl mainly by splicing of multiple exons of the c-rab gene.
TL;DR: Sequence comparison showed a striking homology of exon sequences and sequences up to 215 base pairs upstream of the mRNA start between the mouse and the human alpha 1(I) collagen gene, suggesting that this region has an important role in the control of tissue-specific collagen expression.
Abstract: Integration of the Moloney murine leukemia virus (M-MuLV) into the germ line of Mov-13 mice blocked formation of stable alpha 1(I) collagen mRNA and led to an embryonic lethal mutation. A 14-kilobase fragment representing the integration site of the virus was molecularly cloned and identified as the alpha 1(I) collagen gene. Sequence and nuclease S1 mapping analyses were performed to characterize the position of the proviral genome in relation to the transcriptional map of the mutated gene. The results indicated that the virus has inserted into the first intron 19 base pairs downstream of the intron/exon boundary. Sequence comparison showed a striking homology of exon sequences and sequences up to 215 base pairs upstream of the mRNA start between the mouse and the human alpha 1(I) collagen gene. This indicates that the sequences upstream of the mRNA start are highly conserved during evolution, suggesting that this region has an important role in the control of tissue-specific collagen expression.
TL;DR: One single glucoamylase gene could be identified in the chromosomal DNA of Aspergillus niger by Southern blot analysis by identifying the internal conserved sequence TACTAAC, which is also found in yeast chromosomal gene introns, and is thought to participate in mRNA splicing.
Abstract: One single glucoamylase gene could be identified in the chromosomal DNA of Aspergillus niger by Southern blot analysis. This glucoamylase gene was isolated from a genomic library of A. niger DNA. The glucoamylase gene is situated on a 2.5-kb EcoRI-EcoRV fragment and contains five intervening sequences in the coding region. One 169-bp intron is involved in differential mRNA processing leading to the two different glucoamylase enzymes G1 and G2; the other four introns are all very small ranging from 55 to 75 bp in length. One intron has a significant homology to the coding region which immediately follows, and it contains the internal conserved sequence TACTAAC, which is also found in yeast chromosomal gene introns, and is thought to participate in mRNA splicing. Two transcription initiation sites and a typical eukaryotic promoter region with TATAAT and CAAT boxes are located upstream from the gene.
TL;DR: The structure of the gene encoding rat pre-proglucagon, a polyprotein precursor of glucagon, is reported, which consists of six exons and five introns and contains repetitive sequence DNA.
TL;DR: In vivo, RNA I controls plasmid copy number and incompatibility and inhibits expression of a galK gene fused to the primer promoter and can be explained by alteration of the binding of RNA I to RNA II by the Rom protein.
TL;DR: Mouse hepatitis virus, which replicates in cytoplasm, contains leader RNA sequences at the 5' end of the virus-specific mRNAs that share extensive sequence homology with the long-terminal-repeat region of several mammalian sarcoma viruses, suggesting possible common functions.
Abstract: Mouse hepatitis virus, which replicates in cytoplasm, contains leader RNA sequences at the 5' end of the virus-specific mRNAs. We have sequenced this leader RNA by synthesizing cDNA from a synthetic oligodeoxyribonucleotide primer (15-mer) that is complementary to the sequences at the junction site between the leader and body sequences of the mRNAs. The leader sequences on each mRNA have exactly the same size, which span approximately equal to 70 nucleotides. Leader cDNA fragments obtained from several mRNA species were sequenced and found to be identical. Computer analysis of the leader RNA sequences shows that they share extensive sequence homology with the long-terminal-repeat region of several mammalian sarcoma viruses, suggesting possible common functions. This is a novel case of spliced leader sequences in the mRNAs of a cytoplasmic virus. An identical leader sequence is also present at the 5' end of the virion genomic RNA. The leader RNA is thus probably encoded by the virion genomic RNA template and is fused to the different body sequences of the various mRNAs. Since conventional RNA splicing is not involved, a novel mechanism for fusing two noncontiguous RNA segments in the cytoplasm must be utilized during viral transcription. Several minor cDNA bands longer than the leader were also synthesized, suggesting the possible presence of partially homologous sequences in other parts of the genome RNA.