Splice Consensus Sequence Research Articles

Cloning of the gene coding for a human receptor for formyl peptides. Characterization of a Promoter Region and Evidence for Polymorphic Expression

Recently we reported that, in HL-60 cells, transcription of the formyl peptide receptor (FPR) gene can be up- and downregulated by agents that induce differentiation of HL-60 cells into neutrophils. To begin studying the mechanisms involved in regulation of FPR gene expression, we cloned two human cDNAs and the gene coding for FPR. The genomic clone (pINF14) contained a 14.5-kb insert. A 2.7-kb EcoRI fragment was obtained from pINF14 that hybridized with an FPR open reading frame probe. The EcoRI fragment was sequenced and found to contain an intronless FPR open reading frame. Sequence alignment of the EcoRI genomic fragment with the FPR cDNA revealed that the first 31 bases of 5' untranslated FPR cDNA were not represented in the genomic fragment. Furthermore, a splicing consensus sequence was present in the genomic fragment at the site of divergence with the cDNA sequence. Restriction mapping and Southern blot analysis identified a 121-bp fragment that contained the sequence corresponding to the first 31 bases of 5' untranslated FPR cDNA. An additional (previously undescribed) 15-bp cDNA sequence in the 5' end of FPR were identified using an anchored polymerase chain reaction. This sequence was also contained in the genomic 121-bp fragment. This 121-bp fragment was located 5.2 kb (intron) upstream of the FPR open reading frame. It contained an unusual TATA box and displayed transcriptional activity in vitro and in vivo. Potential binding sites for AP-1 and glucocorticoid receptor were identified upstream of the putative TATA box.(ABSTRACT TRUNCATED AT 250 WORDS)

Enteric adenovirus type 40: E1B transcription map and identification of novel E1A–E1B cotranscripts in lytically infected cells

Adenovirus 40 (Ad40) is defective for growth in tissue culture but is complemented when the Ad2/5 or Ad12 E1B 55K protein is supplied in trans. Ad40 E1B mRNA has not been detected in E1-transformed cells, or at early times in lytically infected cells. In cells constitutively expressing the E1B region of Ad2, Ad40 E1B mRNAs are detected at late times in infection, after the onset of DNA replication. We have determined the Ad40 E1B transcription map from RNA produced at late times in infected KB16 cells, using S1 nuclease, primer extension, PCR-cDNA analysis, and Northern blotting. E1B transcripts corresponding to Ad2 14 S, 22 S, and 9 S mRNAs were identified but no 13 S mRNA equivalent was detected, a pattern similar to that seen in the Adl2 transcription map. The coding potential for E1B 19K, 55K, and 15K proteins and for ppIX is retained in the Ad40 transcripts. In addition we find novel E1A–E1B cotranscript counterparts of the 14 S and 22 S mRNAs. These contain the first 40 codons of the E1A first exon linked to a site 4–5 nt downstream of the E1B cap site, retaining all the coding potential of the E1B mRNAs. No new open reading frames are created by the junction, and the E1A ORF terminates with one codon added after the junction. Each E1A–E1B cotranscript is present in abundance comparable to that of its authentic E1B counterpart. The E1A–E1B junction is unusual in that it does not conform to splice consensus sequences and thus may not be generated by a conventional splicing mechanism.

Genomic organisation of the bovine alpha-S1 casein gene

We report the sequence of the complete bovine alpha-s1 casein gene eludicating for the first time the genomic organization of an alpha-s type casein gene. Extending over 17508 bp the gene is split into 19 exons, ranging in size from 24 bp to 385 bp. Except for the translational stop codon not a single coding triplet of the alpha-s1 reading frame is disrupted by any of the splice junctions, which all confirm to known splice consensus sequences. Nine out of 16 coding exons begin with a 'GAX' codon, specific for glutamate. Splicing of this codon from exon 10 to the preceding exon creates a major phosphorylation site. An intron-exon-intron stretch of 154 bp comprising exons 10 and 13 is found precisely duplicated. Associated with the gene, copies of 8 atriodactyla retroposons are found, 6 of which are interspersed into the sequences of the three longest introns. We discuss the possibility that three functional parts of the gene have been recruited and evolutionary conserved at a time before gene diversification gave rise to the separate evolution of alpha- and beta-type casein-genes.

Drosophila Transposable Elements: Mechanisms of Mutagenesis and Interactions With The Host Genome

Publisher Summary This chapter discusses the transposable elements of Drosophila . The chapter also discusses the mechanisms of mutagenesis and interactions with the host genome. Most spontaneous mutations in yeast and Drosophila are caused by the insertion of transposable elements. There are many examples of transposon-induced mutations in vertebrates and several recent reports have shown de novo insertions of transposable elements resulting in inheritable genetic defects or neoplastic transformation in humans. Therefore, the generation of mutant phenotypes by the insertion of transposable elements may be an evolutionarily general phenomenon that deserves study for both basic and practical considerations. In Drosophila, transposable elements may insert into genes and cause mutant phenotypes by a variety of mechanisms, the nature of which depends on the location of the insertion site with respect to the different structural and functional domains of the affected gene. Transposable elements may contain splice consensus sequences that when inserted into the transcription unit of a gene could result in aberrant processing patterns, or they may contain transcription termination signals that could cause premature termination of transcription of the mutant gene. Transposable elements also carry sequences necessary for transcription initiation—such as enhancers—that could alter the pattern of expression of adjacent genes.

The small nuclear ribonucleoproteins, SmB and B', are products of a single gene

Nucleotide sequencing of HeLa genomic DNA amplified by the polymerase chain reaction revealed 696-bp and 841-bp introns within the gene encoding the proteins SmB and SmB', respectively. Since splice consensus sequences were observed flanking the 145-bp insertion detected in the cDNA encoding SmB, we conclude that SmB and SmB' are derived from alternative splicing of a single gene transcript.

Structure of the human methylmalonyl-CoA mutase ( MUT) locus

The MUT locus encoding the enzyme methylmalonyl-CoA mutase is defective in mut forms of methylmalonic acidemia. This locus has been mapped to chromosome 6p12–21.1. We report cloning and characterization of this locus which comprises 13 exons spanning >35 kb of the genome. The MUT locus exhibits consensus sequences for transcription, splicing, and polyadenylation. The putative promoter region was localized in a CG island 5′ to exon I and was shown to direct expression of a β-galactosidase reporter gene in cultured cells. Of interest is the observation that the first intron occurs within the 5′ untranslated region, and no introns separate the mitochondrial targeting sequences and the mature apoenzyme. An informative HindIII polymorphism was localized within the coding sequence and can be assayed using the polymerase chain reaction. These studies describe the structure of the MUT locus and provide a foundation for characterization of mutations in mut methylmalonic acidemia.

UV Cross-Linking of Polypeptides Associated with 3′-Terminal Exons

Association of nuclear proteins with chimeric vertebrate precursor RNAs containing both polyadenylation signals and an intron was examined by UV cross-linking. One major difference in cross-linking pattern was observed between this chimeric precursor RNA and precursors containing only polyadenylation or splicing signals. The heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptide C cross-linked strongly to sequences downstream of the A addition site in polyadenylation precursor RNA containing only the polyadenylation signal from the simian virus 40 (SV40) late transcription unit. In contrast, the hnRNP C polypeptide cross-linked to chimeric RNA containing the same SV40 late poly(A) cassette very poorly, at a level less than 5% of that observed with the precursor RNA containing just the poly(A) site. Observation that cross-linking of the hnRNP C polypeptide to elements within the SV40 late poly(A) site was altered by the presence of an upstream intron suggests differences in the way nuclear factors associate with poly(A) sites in the presence and absence of an upstream intron. Cross-linking of C polypeptide to chimeric RNA increased with RNAs mutated for splicing or polyadenylation consensus sequences and under reaction conditions (high magnesium) that inhibited polyadenylation. Furthermore, cross-linking of hnRNP C polypeptide to precursors containing just the SV40 late poly(A) site was eliminated in the presence of competing poly(U); polyadenylation, however, was unaffected. Correlation of loss of activity with high levels of hnRNP C polypeptide cross-linking raises questions about the specificity of the interaction between the hnRNP C polypeptide and polyadenylation precursor RNAs in vitro.

UV cross-linking of polypeptides associated with 3'-terminal exons.

Association of nuclear proteins with chimeric vertebrate precursor RNAs containing both polyadenylation signals and an intron was examined by UV cross-linking. One major difference in cross-linking pattern was observed between this chimeric precursor RNA and precursors containing only polyadenylation or splicing signals. The heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptide C cross-linked strongly to sequences downstream of the A addition site in polyadenylation precursor RNA containing only the polyadenylation signal from the simian virus 40 (SV40) late transcription unit. In contrast, the hnRNP C polypeptide cross-linked to chimeric RNA containing the same SV40 late poly(A) cassette very poorly, at a level less than 5% of that observed with the precursor RNA containing just the poly(A) site. Observation that cross-linking of the hnRNP C polypeptide to elements within the SV40 late poly(A) site was altered by the presence of an upstream intron suggests differences in the way nuclear factors associate with poly(A) sites in the presence and absence of an upstream intron. Cross-linking of C polypeptide to chimeric RNA increased with RNAs mutated for splicing or polyadenylation consensus sequences and under reaction conditions (high magnesium) that inhibited polyadenylation. Furthermore, cross-linking of hnRNP C polypeptide to precursors containing just the SV40 late poly(A) site was eliminated in the presence of competing poly(U); polyadenylation, however, was unaffected. Correlation of loss of activity with high levels of hnRNP C polypeptide cross-linking raises questions about the specificity of the interaction between the hnRNP C polypeptide and polyadenylation precursor RNAs in vitro.

Structure and chromosomal location of the human gene encoding cartilage matrix protein.

Cartilage matrix protein (CMP) is a major component of the extracellular matrix of nonarticular cartilage. The structure and chromosomal location of the human gene encoding CMP was determined by molecular cloning analysis. We used a partial chicken CMP cDNA probe to isolate three overlapping human genomic clones. From one of these clones, a probe containing 2 human CMP exons was isolated and used to map the gene to chromosome 1p35 and to screen a human retina cDNA library. Two overlapping cDNA clones were isolated. The predicted protein sequence of 496 amino acids includes a 22-residue signal peptide and a 474-residue mature protein of Mr 51,344. The human CMP gene and polypeptide are strikingly similar to the chicken CMP gene and polypeptide. Human CMP is 79% identical to chicken CMP and contains two homologous domains separated by an epidermal growth factor-like domain. One potential N-glycosylation site is conserved between the two species. The human CMP gene spans 12 kilobase pairs with 8 exons and 7 introns which are similar in size to those of the chicken CMP gene. Both RNA splice junctions of intron G in the human and chicken CMP genes are nonconforming to the consensus splice sequences. This suggests that the CMP gene utilizes a new RNA splicing mechanism.

Human Nuclear NAD+ADP-Ribosyltransferase(polymerizing): Organization of the Gene

Human nuclear NAD+: protein ADP-ribosyltransferase(polymerizing) [pADPRT; poly(ADP-ribose)poly-merase; EC 2.4.2.30] is a DNA-dependent protein-modifying enzyme composed of several domains important for DNA binding, automodification, and NAD binding. We report that the human pADPRT gene is 43 kb in length and is split into 23 exons. All the intron-exon boundaries correspond to a canonical splice consensus sequence. Each of the four metal coordinating sites putatively forming the two zinc fingers of the DNA-binding domain is encoded separately. The automodification domain and the NAD-binding domain are coded for by 4 and 12 exons, respectively.

Abnormal processing of β-Malay globin RNA

Hemoglobin Malay (α 2β 219Asn→Ser) has been observed in a few Malaysian patients with thalassemia intermedia. The β Malay substitution increases the homology of the cryptic splice site at codons 17/18/19 of the β-globin gene to the donor consensus splice sequence, suggesting that the β-thalassemia associated with this mutation may be due to the generation of a new splice site. To test this hypothesis, we constructed a hybrid gene where we replaced part of a normal β-globin gene with a PCR amplified region of the β Malay gene. The expression of this mutant gene was studied in a heterologous transient expression system. The data show that nearly 25% of globin mRNA produced by this gene is abnormally spliced at the new splice site, providing a molecular mechanism for the β-thalassemia associated with the mutation.

Structure of the gene encoding the human B lymphocyte differentiation antigen CD20 (B1).

The CD20 (B1) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In this report genomic DNA clones containing the human CD20 gene were isolated and the structure of the CD20 gene determined. Southern blot analysis revealed that CD20 mRNA was transcribed from a single-copy gene. The CD20 gene was 16 kb long and was composed of eight exons. The first exon marked the major transcription initiation site as determined by primer extension and S1 nuclease analysis. The translation initiation codon was located within the third exon. Exon VIII encoded the COOH terminus of the CD20 protein and the long 3' untranslated region. Three forms of CD20 mRNA were identified that all encode an identical protein product. The dominant form of 2.8 kb results from usage of exons I through VIII, whereas a second form that is 263 bp shorter had exon I spliced into an internal 3' splice site within exon III thereby skipping exon II. A minor 3.4-kb mRNA species most likely results from an uncharacterized upstream exon(s) splicing into an internal 3' slice site located in exon I. Nucleotide sequences of cDNA clones representative of each of these RNA forms are presented. The 5' splice site following exon V was found to be divergent from the consensus splice sequence. A relationship between the individual peptides encoded by the six exons and structurally distinct regions of the CD20 protein is likely.

Organization and expression of the human myelin basic protein gene

The human brain contains four isoforms of myelin basic protein (MBP), previously identified by cDNA cloning. We have now isolated and characterized genomic clones encoding the human MBP gene. The gene is 45 kb in extent and consists of seven exons. Alternative splicing of the primary MBP transcript can account for all four human MBP isoforms. The intron-exon boundaries of the gene have also been determined, and all conform to the known consensus splice sequences. These sequences, however, do not explain the alternative splicing pattern found in human brain. Transcription of the human MBP gene begins at a single site within the MBP promoter, and all four MBP isoforms are transcribed from this same site. The promoter region does not contain any known sequence elements, but does have a 12-bp sequence also found in the JC virus 98-bp tandem repeat. A relative gradient of MBP transcription is found from caudal to rostral within the developing human brain, which parallels the known sequence of myelination found in these areas. RNase protection of brain RNA demonstrates more of the 21.5-kD and 20.5-kD MBP mRNAs in neonatal brain than in the adult frontal cortex, which suggests that alternative splicing of the primary MBP transcript is also regulated temporally during myelin development. These data show that regulation of myelination is complex, involving regional cellular interactions and trans activation of transcription, as well as modulation of alternative splicing. Comparison of the human and mouse data also suggests that alternative splicing plays an important role in myelin biogenesis.

Maturation of the major ubiquitin gene transcript in Caenorhabditis elegans involves the acquisition of a trans-spliced leader.

Recently, studies on the 5'-ends of actin mRNAs in the nematode, Caenorhabditis elegans, have demonstrated that three of the four mature actin transcripts contain a 22-nucleotide leader sequence which is acquired by trans-splicing from a novel 100-nucleotide RNA. In the course of our studies of the ubiquitin genes in C. elegans (R. W. Graham and E. P. M. Candido, manuscript in preparation) we were led to the possibility that the major ubiquitin transcript in this organism might also contain a trans-spliced leader sequence. The 5'-noncoding region of the major ubiquitin gene (UbiA) contains a 3'-splice consensus sequence six nucleotides upstream of the initiation codon; however, sequencing of a further 1.6 kilobases upstream failed to reveal the existence of any potential 5'-splice sites in the correct relationship to known promoter elements. Furthermore, S1 and Northern blot analyses using probes complementary to upstream regions failed to detect the ubiquitin transcript. Primer extension experiments on total RNA using an oligonucleotide which hybridized to the ubiquitin transcript downstream of the 3'-splice site demonstrated the existence of a 22-nucleotide leader sequence not present in the ubiquitin gene 5'-region. Thus we show here that the 2500-nucleotide mRNA encoding a polyubiquitin precursor protein contains a 22-nucleotide leader sequence identical to that found in several C. elegans actin mRNAs and which is most likely acquired by a trans-splicing mechanism. In addition we have localized the site of transcript initiation for UbiA by S1 nuclease mapping.

Antibodies with specificities related to the Kell blood group system

The 22 antigens of the Kell blood group system are located on a red blood cell (RBC) membrane glycoprotein that shows sequence homology with a family of metalloendopeptidases. Expression of the Kell system antigens is partially governed by XK, an X-linked gene that encodes the Kx protein; absence of Kx results in reduced Kell antigen expression. Almost total absence of Kell antigens from the RBCs of a German man with no symptoms of neuroacanthocytosis could not be due to the Kell-null phenotype, K0, because his RBCs had very weak expression of Kx antigen and his three children were Kp(a + b+). Kell antigens were normal on the RBCs of his son but weak on those of his two daughters. An Nla III restriction fragment-length polymorphism within the KEL gene showed the Kpa/Kpa genotype in the propositus. Sequencing of his XK gene showed a single base change within the donor splice consensus sequence of intron 2. A BsaAl restriction fragment-length polymorphism showed the mutation in both of his daughters but not in his son. The extreme depression of the Kell antigens of the propositus must be due to a combination of effects, ie, homozygosity for Kpa and deficiency of Kx protein, each of which is capable of causing some degree of weakening of Kell antigens.

Human myelin DM-20 proteolipid protein deletion defined by cDNA sequence

Using a myelin proteolipid protein (PLP) cDNA as probe, a 1kb cDNA was isolated from a human retinal cDNA library. This clone, designated pDM-20, contained all of the coding sequence of PLP except for 105 base pairs which encode amino acids 117-151 of PLP. This deletion would result in a protein of the size of DM-20, the second major proteolipid of Central Nervous System (CNS) myelin. Homology between the DNA sequence at the 5' end of the deletion and the donor splicing consensus sequence suggests that PLP and DM-20 transcripts are derived from a single PLP gene by alternate splicing.

Structure and developmentally regulated expression of a Strongylocentrotus purpuratus collagen gene.

We have isolated and partially characterized an ca. 20-kilobase-pair Strongylocentrotus purpuratus genomic clone, using a mouse alpha 1 (type IV) collagen cDNA probe. A 1-kilobase-pair HindIII fragment of the clone hybridizes strongly to the probe; this has been subcloned and sequenced. It contains 212 base pairs of sequence coding for (Gly-Xaa-Yaa)n (where Xaa and Yaa are different unspecified amino acids), characteristic of all known collagen genes. There is a single point of discontinuity within the repeating pattern in this exon, similar to the genomic structure of mouse type IV collagen. The (Gly-Xaa-Yaa)n-encoding element is flanked by consensus splicing sequences, and the intervening sequences on either side of it have multiple in-phase termination codons. Electron microscopy of R loops between the phage lambda recombinant clone and poly(A)+ RNA reveals multiple short exons, a feature also seen in vertebrate collagen genes. The (Gly-Xaa-Yaa)n protein-encoding sequence hybridizes to a developmentally regulated 9-kilobase mRNA; the message appears during the morula stage, rises sharply in abundance at the blastula stage, and decreases in proportion to total RNA later in development.

Site-specific deletion in cauliflower mosaic virus DNA: possible involvement of RNA splicing and reverse transcription

A frequent site-specific deletion was observed in the life cycle of cauliflower mosaic virus (S strain). Analysis of the sequence around the deletion site and the parental sequence implied that the deletion was promoted at sequences similar to the donor and acceptor consensus sequences of RNA splicing, designated as the deletion donor and acceptor sequences, respectively. To elucidate the mechanism of this site-specific deletion, point mutations were introduced into the deletion donor sequence (GT to GG or GA transversion). Deletion at the original deletion donor site did not occur in these mutants, instead, new (cryptic) donor sites were activated. All of these activated cryptic sites had sequences similar to the splicing consensus sequence. In all cases except one, the original deletion acceptor site was used. These results can be most readily explained by postulating that the site-specific deletion occurs by reverse transcription of spliced viral RNA. This frequent site-specific deletion was not observed in other strains. For a virus which replicates by reverse transcription, a mechanism to regulate the rate of splicing is required to ensure the intactness of the viral genome. We discuss the possibility that the S strain has a mutation in this regulatory mechanism.

The complete nucleotide sequence of the Neurospora crassa am (NADP-specific glutamate dehydrogenase) gene

The complete nucleotide sequence of a 2.7-kb genomic fragment, containing the Neurospora crassa am [NADP-specific glutamate dehydrogenase (GDH)] gene, has been determined. The transcription initiation and polyadenylation sites have been defined by S1 mapping. There are at least four initiation sites between 35 and 60 bases downstream of a TATAAA sequence. The single polyadenylation site is immediately downstream of a six-nucleotide sequence which is present in the corresponding position in the N. crassa gene ( pl) coding for the imported proteolipid component of mitochondrial ATP synthase [Viebrock et al. Embo J. 1 (1982) 565–571] the initiation sites also show some sequence similarity between am and pl. There are two introns, of 66 and 61 bases respectively; they interrupt sequences that are conserved (highly so in the case of the second intron) between Neurospora and Escherichia coli. As well as showing typical consensus splicing sequences, the two introns show some striking internal similarities to each other. The am gene resembles the pl gene in its strongly selective use of codons (pyrimidines preferred to purines and guanine to adenine in third positions). The coding sequence here reported requires four corrections to be made in the previously published amino acid sequence of Neurospora GDH.

Complete sequence analyses show that two defective interfering influenza viral RNAs contain a single internal deletion of a polymerase gene.

Defective interfering (DI) influenza viral RNAs arise by internal deletion of progenitor RNAs. By using recombinant DNA cloning and DNA sequence analysis techniques, we have deduced the complete sequence of two such RNAs (L2b and L3), both arising from the same polymerase (P1) gene of WSN influenza virus. We have also partially determined the sequence of the P1 polymerase gene, including the sequence at the point of deletion and the flanking regions. Our sequence study shows the following. (i) Both L2b and L3 arise by a simple deletion in the P1 gene. (ii) L2b and L3 are 683 and 441 nucleotides long, respectively. (iii) The first 413 and 244 nucleotides of the 5' ends of L2b and L3, respectively, are identical to those of the 5' end of the P1 gene. (iv) The last 270 nucleotides of L2b and 197 nucleotides of L3 are the same as those of the 3' end of the P1 gene. (v) The entire sequence of L3 is present in the sequence of L2b. (vi) Both the 5' and the 3' termini, including the transcription stop and poly(A) addition signals of the progenitor P1 gene, are present in both L2b and L3. (vii) The sequences at the deletion point and the flanking region of the P1 gene do not resemble the consensus splicing sequence of spliced mRNA suggesting that a replicational event rather than splicing is involved in the formation of influenza defective interfering RNAs.