Showing papers on "Intron published in 1989"

Alternative splicing directs the expression of two D2 dopamine receptor isoforms

Abstract: Dopamine receptors are classified into D1 and D2 subtypes on the basis of their pharmacological properties and the intracellular responses they mediate. The cerebral D2 dopamine receptor is the target of drugs used to alleviate the main symptoms of schizophrenia. Although it is considered to be a single molecular entity, there is evidence that multiple D2-receptor subtypes exist. A complementary DNA encoding a D2 receptor has recently been cloned and the deduced 415-amino-acid sequence indicates that it belongs to the large superfamily of receptors coupled to G proteins, and that its topology consists of seven transmembrane domains. In this family, the genes are frequently without introns and each is believed to encode a unique polypeptide product. Here we show that the gene for the D2 receptor produces two receptor isoforms by alternative messenger RNA splicing, providing a route to receptor diversity in this family. One isoform corresponds to the D2(415) receptor, but the second contains an additional sequence encoding a 29-amino-acid fragment, defining a novel D2(444) receptor isoform. Expression of the two isoforms is tissue-specific, and both are regulated by guanyl nucleotides. As the extra sequence is located within a putative cytoplasmic loop that binds to G proteins, the two isoforms might interact with different G proteins and thereby initiate distinct intracellular signals.

Alternative splicing in the control of gene expression

Abstract: In this review, we focus upon the mechanistic, functional, and evolutionary aspects of alternative splicing. The discussion of mechanisms attempts to be exhaustive, drawing not only upon direct experimental investigations of alternatively spliced genes, but also upon relevant themes derived from the study of constitutive splicing

Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8.

Abstract: To identify possible regulatory sites of the gene expression, we cloned the genomic DNA of human monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8), whose mRNA is induced by IL-1 or TNF and determined its entire nucleotide sequence. The results show that the MDNCF/IL-8 gene consists of 4 exons and 3 introns with a single "CAT"- and "TATA"-like structure. The 5'-flanking region of MDNCF/IL-8 gene shows no overall sequence similarity with that of other cytokine and acute phase reactant genes whose production is also affected by IL-1 and TNF. The 5' flanking region, however, contains potential binding sites for several nuclear factors including activation factor-1, activation factor-2, IFN regulatory factor-1, and hepatocyte nuclear factor-1. In addition, the glucocorticoid responsive element and heat shock element were located in the 5'-flanking region. Inasmuch as PMA induces MDNCF/IL-8 mRNA accumulation in human PBMC and a glucocorticoid inhibits the induction of MDNCF/IL-8 mRNA by LPS, the expression of this gene is probably regulated by interaction of these nuclear factors with the 5'-flanking DNA.

Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity.

Abstract: Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. We have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46,XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development.

RNA editing in wheat mitochondria results in the conservation of protein sequences.

Abstract: RNA editing is a process that results in the production of a messenger RNA with nucleotide sequences that differ from those of the template DNA1, and provides another mechanism for modulating gene expression. The phenomenon was initially described in the mitochondria of protozoa2, 3. Here we report that RNA editing is also required for the correct expression of plant mitochondria! genes. It has previously been proposed that in plant mitochondria there is a departure from the universal genetic code4, with CGG specifying tryptophan instead of arginine. This was because CGG codons are often found in plant mitochondrial genes at positions corresponding to those encoding conserved tryptophans in other organisms. We have now found, however, wheat mitochondrial gene sequences containing C residues that are edited to U residues in the corresponding mRNA sequences. In this way, CGG codons can be changed to UGG codons in the mRNA so that tryptophan may be encoded according to the universal genetic code. Furthermore, for each codon modification resulting from a C - > U conversion that we studied, we found a corresponding change in the amino acid that was encoded. RNA editing in wheat mitochondria can thus maintain genetic information at the RNA level and as a result contribute to the conservation of mitochondrial protein sequences among plants.

Characterization of the glycinin gene family in soybean.

Abstract: We characterized the structure, organization, and expression of genes that encode the soybean glycinins, a family of storage proteins synthesized exclusively in seeds during embryogenesis. Five genes encode the predominant glycinin subunits found in soybeans, and they have each been cloned, sequenced, and compared. The five genes have diverged into two subfamilies that are designated as Group-I and Group-II glycinin genes. Each glycinin gene contains four exons and three introns like genes that encode related proteins in other legumes. Two other genes have been identified and designated as "glycinin-related" because they hybridize weakly with the five glycinin genes. Although not yet characterized, glycinin-related genes could encode other glycinin subunit families whose members accumulate in minor amounts in seeds. The three Group-I glycinin genes are organized into two chromosomal domains, each about 45 kilobase pairs in length. The two domains have a high degree of homology, and contain at least five genes each that are expressed either in embryos or in mature plant leaves. Gel blot studies with embryo mRNA, as well as transcription studies with 32P-RNA synthesized in vitro from purified embryo nuclei, indicate that glycinin and glycinin-related genes become transcriptionally activated in a coordinated fashion early in embryogenesis, and are repressed coordinately late in seed development. In addition to transcriptional control processes, posttranscriptional events also are involved in regulating glycinin and glycinin-related mRNA levels during embryogenesis.

Structure of the rat osteocalcin gene and regulation of vitamin D-dependent expression.

Abstract: The osteocalcin gene encodes a 6-kDa polypeptide, which represents one of the most abundant noncollagenous bone proteins, and the present studies establish that osteocalcin mRNA is detected only in bone tissue. An osteocalcin gene was isolated from a rat genomic DNA library, and sequence analysis indicated that the mRNA is represented in a 953-nucleotide segment of DNA consisting of four exons and three introns. A modular organization of the 5' flanking sequences of the gene is reflected by the presence of at least three classes of regulatory elements, which include the following: (i) RNA polymerase II canonical sequences; (ii) a series of consensus sequences for hormone receptor binding sites and cyclic nucleotide responsive elements consistent with physiologic expression of the osteocalcin gene; and (iii) a 24-nucleotide sequence in the proximal promoter region with a CAAT motif as a central element. We have designated this highly conserved sequence as an "osteocalcin box" since only 2 nucleotide substitutions are found in the rat and human osteocalcin genes. We have demonstrated two factors regulating osteocalcin gene expression. First, a 200-fold increase occurs in normal fetal calvaria osteoblasts producing a mineralizing matrix, compared to confluent osteoblasts in a nonmineralizing matrix. Second, contained within the 600 nucleotides immediately upstream from the transcription start site are sequences that support a 10-fold stimulated transcription of the gene by 1,25-dihydroxyvitamin D.

Structure of the human insulin receptor gene and characterization of its promoter

Abstract: The human insulin receptor gene, INSR, and its promoter region have been isolated and characterized. The gene spans greater than 120 kilobase pairs (kbp) and has 22 exons. All introns interrupt protein coding regions of the gene. The 11 exons encoding the alpha subunit of the receptor are dispersed over greater than 90 kbp, whereas the 11 exons encoding the beta subunit are located together in a region of approximately 30 kbp. Three transcriptional initiation sites have been identified and are located 276, 282, and 283 bp upstream of the translation initiation site. In addition, a 247-bp fragment from the promoter region possessing 62.6% of the maximal promoter activity has been identified. This promoter-active fragment lacks a TATA-like sequence but has two possible binding regions for the transcriptional factor Sp1. Comparison of the exon structure of the tyrosine kinase domain of the INSR with the corresponding regions of the human SRC, ROS, and ERBB2 (NGL) protooncogenes indicates that the exon-intron organization of this region has not been well conserved.

Expression of the murine alpha B-crystallin gene is not restricted to the lens.

Abstract: The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.

Isolation and expression of an anther-specific gene from tomato.

Abstract: We have isolated and sequenced an anther-specific cDNA clone and a corresponding genomic clone from tomato. The gene (LAT52) encodes an 800-nucleotide-long transcript that is detectable in pollen, anthers and at 20- to 50-fold lower levels in petals. LAT52 mRNA is not detectable in pistils, sepals or non-reproductive tissues. Steady-state levels of LAT52 mRNA are detectable in immature anthers containing pollen at the tetrad stage and increase progressively throughout microsporogenesis until anthesis (pollen shed). The LAT52 gene contains 5' and 3' untranslated regions of 110 and approximately 150 nucleotides, respectively, and a single intron with a highly repetitive sequence. A TATA box motif is located 28 nucleotides upstream of the transcription start site. The gene encodes a putative protein of 18 kDa that is cysteine rich and has an N-terminal hydrophobic region with characteristics similar to eucaryotic secretory signal sequences. LAT52 is a single or low copy gene in tomato and shares homology with sequences in tobacco.

Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing.

Abstract: SV40 early pre-mRNA is alternatively spliced to produce large T and small t mRNAs by use of different 5'-splice sites and a shared 3'-splice site. The large T splicing pathway uses multiple lariat branch sites, whereas small t splicing, constrained by its small intron size, can use only one. We exploited this situation to test the hypothesis that RNA-RNA base pairing between U2 snRNA and the branch site sequence is important in mammalian pre-mRNA splicing by constructing and analyzing several mutations in the small t pre-mRNA branch site (UUCUAAU). All of the mutations resulted in substantial decreases in small t splicing relative to large T. To test whether these effects resulted from decreased base pairing with U2 snRNA, compensatory mutations were introduced at the appropriate positions (nucleotides 34-36) in a cloned human U2 gene. All branch site mutations tested (four separate single base substitutions and two triple mutations) were suppressed (i.e., small t splicing was increased) by the appropriate U2 mutations. These results establish that recognition of the poorly conserved mammalian pre-mRNA branch site sequence by U2 snRNP can involve base-pairing.

Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae.

Abstract: In this study we report the isolation of temperature-sensitive mutants that affect pre-mRNA splicing. A bank of ~1000 temperature-sensitive Saccharomyces cerevisiae strains was generated and screened on RNA gel blots by hybridization with an actin intron probe. We isolated 16 mutants defining 11 new complementation groups prp(ma)17-prp(ma}27 with four phenotypic classes of mutants and 21 mutants in the prp2-prpll complementation groups (formerly ma2-mall]. The majority of the complementation groups share a phenotype of pre-mRNA accumulation, seen in all of the prp(ma}2-prp(ma)ll mutants. Three novel classes of mutants were isolated in this study. One class, consisting of two complementation groups, exhibits an accumulation of the lariat intermediate oi splicing, with no change in the levels of pre-mRNA. The second class, also represented by two complementation groups, shows an accumulation of the intron released after splicing. The third novel class, comprising one complementation group, accumulates both pre-mRNA and the released intron. All mutants isolated were recessive for the splicing phenotype. Only 2 of the 11 complementation groups, although recessive, were not temperature sensitive. This study, together with previous isolation of the prp(ma)2-prp(ma)ll groups and the spliceosomal snRNAs, puts at least 26 gene products involved directly or indirectly in pre-mRNA splicing.

Definition of an efficient synthetic poly(A) site.

Abstract: We constructed and analyzed a synthetic poly(A) (SPA) site that was based on the highly efficient poly(A) signal of the rabbit beta-globin gene. By use of the SPA, we demonstrate that the minimum sequences required for efficient polyadenylation are the AATAAA sequence and a GT/T-rich sequence with the correct spacing of 22-23 nucleotides between them. When placed downstream of the poly(A) site of the human alpha 2-globin gene, the SPA is used exclusively. We predict that the SPA, with its more extensive GT/T-rich sequence, is a more efficient poly(A) site than alpha-globin. Also, we compared the use of the SPA when it is placed either in the exon 3 or intron 2 of the rabbit beta-globin gene. When in the exonic position, SPA is used 10-fold more than the regular poly(A) site of rabbit beta-globin. In contrast, when it is in the intronic location, no detectable use of SPA is observed; however, the deletion of the donor site of intron 2 reactivates the intronic positioned SPA. These results indicate that the splicing of intron 2 in the rabbit beta-globin gene occurs ahead of polyadenylation and have important implications for termination of transcription. Polyadenylation, although required for termination of transcription, is not sufficient; therefore, additional termination signals for RNA polymerase II must exist.

Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms.

Abstract: A new promoter located within E6 was mapped in human papillomavirus type 6b (HPV6b)- and HPV11-containing benign genital condylomata (genital warts). The RNA transcribed from this promoter represented the major RNA species colinear with open reading frames E6 and E7 and can encode the E7 protein. No equivalent promoter was active in HPV16-containing cancers and cancer-derived cell lines. In those, the major transcripts contained one of two different introns within E6 and the RNAs could encode two different E6 proteins and E7.

Two classes of CD1 genes

Abstract: Herein, we report the DNA sequence of two human CD1 genes, R2 and R3, distinct from those encoding the CD1a, -b and -c antigens. Both genes appear to have an exon/intron structure analogous to the previously analyzed CD1 genes and to be functional on the basis of their sequence. Analysis of the variability patterns, potential intramolecular interactions and predicted secondary structure profile on an alignment of all known CD1 α chains suggest some shared structural features with major histocompatibility complex class I molecules in the α1 domains but substantial differences in the α2 domains. Sequence comparison shows that, while R2 is most related to CD1a, -b and -c, albeit to a somewhat lower degree than the latter are to themselves, R3 is more homologous to mouse than to human CD1, suggesting the existence of two functional classes within the CD1 gene family. We propose to retain the non-committal R2 and R3 names until the putative antigens have been identified and their tissue distribution has been established.

Scanning from an independently specified branch point defines the 3' splice site of mammalian introns

Abstract: During pre-messenger RNA splicing the lariat branch point in mammalian introns is specified independently of the 3' splice site by the sequence surrounding the branch point and by an adjacent downstream polypyrimidine tract. The 3' splice site is dispensable for spliceosome assembly and cleavage at the 5' splice site, and is itself determined by a scanning process that recognizes the first AG located 3' of the branch point/polypyrimidine tract, irrespective of distance or sequence environment.

Gene Structure, Organization, And Expression In Archaebacteria

Abstract: Major advances have recently been made in understanding the molecular biology of the archaebacteria. In this review, we compare the structure of protein and stable RNA-encoding genes cloned and sequenced from each of the major classes of archaebacteria: the methanogens, extreme halophiles, and acid thermophiles. Protein-encoding genes, including some encoding proteins directly involved in methanogenesis and photoautotrophy, are analyzed on the basis of gene organization and structure, transcriptional control signals, codon usage, and evolutionary conservation. Stable RNA-encoding genes are compared for gene organization and structure, transcriptional signals, and processing events involved in RNA maturation, including intron removal. Comparisons of archaebacterial structures and regulatory systems are made with their eubacterial and eukaryotic homologs.