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PubMed1998

Code growth, explicitly defined introns, and alternative selection schemes.

Previous work on introns and code growth in genetic programming is expanded on and tested experimentally. Explicitly defined introns are introduced to tree-based representations as an aid to measuring and evaluating intron behavior. Although it is shown that introns do create code growth, they are not its only cause. Removing introns merely decreases the growth rate; it does not eliminate it. By systematically negating various forms of intron behavior, a deeper understanding of the causes of code growth is obtained, leading to the development of a system that keeps unnecessary bloat to a minimum. Alternative selection schemes and recombination operators are examined and improvements demonstrated over the standard selection methods in terms of both performance and parsimony.

Biological Evolution↗
PubMed1999

Involvement of intronic sequences in cell-specific expression of the peripherin gene.

Peripherin is an intermediate filament protein expressed in restricted populations of neurons. Our previous study of the chromatin structure of the mouse peripherin gene in cells that do or do not express peripherin suggested that the region located between -1,500 and +800 bp of the gene could be involved in its cell specificity. In the present work, we performed an in vitro functional analysis of the 5' flanking region of the mouse peripherin gene and observed that this region up to 9 kb contained both enhancer and inhibiting activities; however, it was insufficient to achieve a complete extinction of reporter gene expression in peripherin-negative cells. Furthermore, analysis of the first three introns with the 5' flanking sequences of the gene showed that intron I greatly increased specificity of the gene expression. Intron I also conferred the same properties to thymidine kinase heterologous promoter. DNase I footprinting experiments performed with intron I revealed at least two protected regions (Inl A and Inl B). Inl A encompasses an AP-2-like binding site that interacted with both neuroblast and fibroblast nuclear factors, as well as with the recombinant AP-2alpha protein. However, gel shift experiments suggested that the interacting nuclear factors are distinct from AP-2alpha itself and probably belong to the AP-2 family. Inl B perfectly matched the consensus binding site for Sp1 and specifically interacted with nuclear protein factors that showed the same binding properties as the Sp1 family members. Fine deletion analysis of intron I indicated that the Inl A element alone is responsible for its enhancing properties, whereas a region located between +789 and +832 gives to intron I its silencer activity.

3T3 Cells↗
PubMed2000

Elevated frequency and functional activity of a specific germ-line p53 intron mutation in familial breast cancer.

Previous studies have determined that the frequency of germ-line p53 mutations in familial breast cancer patients is 1% or less, but these reports have not investigated the importance of polymorphic intron base changes in the p53 gene. Therefore, we investigated the frequency of both exon and intron germ-line p53 base changes in 42 breast cancer patients with a strong family history of breast cancer. The mean age of presentation of these patients was 44.0 years (range, 29-69), and 12 of 42 (29%) were of known Ashkenazi ancestry. Purified DNA obtained from the 42 index cases was screened for germ-line p53 mutations in exons 2-11 and surrounding introns using a combination of intron based primers for PCR-single strand conformation polymorphism analysis, direct sequencing, and microarray sequencing using the Affymetrix p53 gene chip methodology. Morphological analysis of apoptosis and cell survival determination were performed on EBV-immortalized lymphoblastoid cell lines from two patients with the p53 intron 6 mutation. A germ-line mutation in the p53 gene at nucleotide 13964 with a G to C base change (13964GC) was identified in 3 of 42 (7.1%) hereditary breast cancer patients. Two patients were heterozygous for this mutation, and one patient had a homozygous mutation. In comparison, 0 of 171 (0%) of sporadic breast cancer patients had the p53 13964GC mutation (P = 0.0003). We found that 0 of 42 (0%) of these hereditary breast cancer patients had other germ-line p53 mutation in exons 2-11. However, pedigree analysis demonstrated that all three patients had strong family histories of multiple types of cancers consistent with Li-Fraumeni syndrome but with late age of onset. Comprehensive BRCA1 and BRCA2 nucleotide analysis from patients with the p53 13964GC mutation revealed no concomitant deleterious BRCA1 or BRCA2 mutations, although they were found in the other hereditary breast cancer patients. Functional analysis of two immortalized lymphoblastoid cell lines derived from patients with the p53 13964GC mutation demonstrated prolonged in vitro survival in response to cisplatinum treatment and showed decreased chemotherapy-induced apoptosis. Immunohistochemical analysis of breast tumors from these patients revealed high levels of mutant p53 protein, suggesting a functional mutation in the p53 gene. In summary, we have identified a single p53 intron mutation in familial breast cancer patients that is present at elevated frequency and has functional activity.

Adult↗
PubMed2001

Alternative splicing of U12-dependent introns in vivo responds to purine-rich enhancers.

Alternative splicing increases the coding capacity of genes through the production of multiple protein isoforms by the conditional use of splice sites and exons. Many alternative splice sites are regulated by the presence of purine-rich splicing enhancer elements (ESEs) located in the downstream exon. Although the role of ESEs in alternative splicing of the major class U2-dependent introns is well established, no alternatively spliced minor class U12-dependent introns have so far been described. Although in vitro studies have shown that ESEs can stimulate splicing of individual U12-dependent introns, there is no direct evidence that the U12-dependent splicing system can respond to ESEs in vivo. To investigate the ability of U12-dependent introns to use alternative splice sites and to respond to ESEs in an in vivo context, we have constructed two sets of artificial minigenes with alternative splicing pathways and evaluated the effects of ESEs on their alternative splicing patterns. In minigenes with alternative U12-dependent 3' splice sites, a purine-rich ESE promotes splicing to the immediately upstream 3' splice site. As a control, a mutant ESE has no stimulatory effect. In minigene constructs with two adjacent U12-dependent introns, the predominant in vivo splicing pattern results in the skipping of the internal exon. Insertion of a purine-rich ESE into the internal exon promotes the inclusion of the internal exon. These results show that U12-dependent introns can participate in alternative splicing pathways and that U12-dependent splice sites can respond to enhancer elements in vivo.

Alternative Splicing↗
PubMed2002

Intron 1 is required for cell type-specific, but not injury-responsive, peripherin gene expression.

The "primitive" neurons of the peripheral nervous system (PNS) have the remarkable ability to regenerate new fibers. This regenerative process requires a sequence of gene activation and repression that is poorly understood. One gene that is almost exclusively expressed in neurons of the PNS and is activated after nerve injury is the peripherin intermediate filament gene, but little is known about the genomic elements that control either its restricted expression or its response to nerve injury in adult mice. Previous studies suggested that both 5' flanking sequence and intragenic regions were required for cell type-specific and injury-specific expression. To determine which intragenic regions were critical, mice were generated that expressed peripherin transgenes lacking different introns. Analyses of these mice revealed that deletion of introns 2-8 had no effect on either the cell type-specific or injury-specific expression of the peripherin gene; however, the remaining intron, intron 1, differentially bound Sp1 transcription-related proteins/protein complexes in extracts from peripherin-expressing and nonexpressing tissues. Furthermore, a transgene that lacked intron 1 was not expressed in many neurons that contain endogenous peripherin but was activated after injury. Thus, accurate cell type-specific peripherin gene expression in the PNS depends on elements within intron 1, but other sequences, most likely in the 5'flanking region, are required for activating the peripherin gene in response to nerve injury.

3' Flanking Region↗
PubMed1992

[Characteristics of a positive regulatory element in the first intron of the mts1 gene].

The first intron of the mts1 gene, a gene which is selectively expressed in metastatic cells and in normal cells that are motile, was found to be highly homologous to the CD3 delta enhancer element. Because of the homology between the CD3 delta enhancer and the first intron of mts1, we analysed the first intron of the mts1 gene to determine whether it functions as a transcriptional regulatory element. Highly metastatic CSML-0 cells transfected with chloramphenicol acetyltransferase containing plasmids demonstrated the ability of the mts1 first intron to function as a positive regulatory element. In vitro footprinting analysis using extracts from CSML-0 cells (which express mts1 at low levels) of CSML-100 cells (which express mts1 at high levels) identified a protected 16 nucleotide element in the first intron of mts1, regardless of the extract used. However, in vivo footprinting analysis of the same region identified the protected 16 nucleotide fragment only in the mts1 intron from CSML-100 cells, not from CSML-0 cells. Differences in the methylation pattern of the mts1 gene in CSML-100 cells and CSML-0 cells are known to exist, and may in part be responsible for the mts1 footprinting differences observed in vivo from the different cells lines.

Animals↗
PubMed1992

[Human immunoglobulin heavy-chain gene intronic enhancer segment HE2 (microB) functions as a major B-cell specific cis-element at cellular level as well as in transgenic mice].

Immunoglobulin heavy-chain (IgH) gene expression is regulated in a large part of the IgH gene intronic enhancer which is composed of certain protein-binding motifs. These motifs act as a DNA element which is important for the regulation of the IgH gene transcription. Two of these motifs, HE2 and E6 in the human IgH gene intronic enhancer, are considered to affect the B cell-specific gene expression. To examine the true function of the HE2 and E6 motifs in vivo, we established transgenic mouse lines. A 3'deletion mutant of the IgH gene intronic enhancer which includes an active HE2 motif showed almost the same degree of transcriptional activity and tissue-specificity as the whole IgH gene intronic enhancer. Still more, using the luciferase-assay, it was confirmed at cellular level that the HE2 motif plays an important role in the tissue-specific activity of the IgH gene intronic enhancer. On the other hand, the E6 motif was unable to sufficiently produce the B cell-specific expression in vivo and showed enhancer activity in some non-B cells. In transgenic mice, the IgH gene intronic enhancer functioned not only in B-lymphocytes but also in chorid plexus cells which are similar to glial cells. This suggests the existence of common gene expression control mechanisms between the immune system and the nervous system.

Animals↗
PubMed2005

Endothelial nitric oxide synthase intron 4 polymorphism is a marker for coronary artery disease in African-American and Caucasian men.

OBJECTIVES: We investigated the association of the intron 4 polymorphism of the endothelial nitric oxide synthase (eNOS) gene with coronary artery disease (CAD). BACKGROUND: Genetic alterations in the gene encoding for eNOS could contribute to the development and progression of CAD. METHODS: We genotyped for the eNOS intron 4 polymorphism in 194 subjects undergoing coronary angiography. Genotyping was performed with polymerase chain reaction-restriction fragment length polymorphism for the variable number of tandem repeats in intron 4. Coronary artery disease (CAD) was assessed by quantitative coronary angiography, and endothelial function was measured by brachial ultrasonography. We performed logistic regression analysis for the effect of eNOS intron 4 polymorphism and other coronary risk factors on multi-vessel CAD and endothelial function. RESULTS: The 4a-allele frequency in African Americans was 0.31, while the 4a-allele frequency in Caucasians was 0.15 (P<.001). The prevalence of the 4a-allele was highest among subjects with multi-vessel disease both for African Americans and for Caucasians. A race-adjusted comparison of the prevalence of the 4a-allele among subjects with multi-vessel disease vs those without was statistically significant (P=.03). No correlation was found between the eNOS intron 4 polymorphism and endothelial function. CONCLUSIONS: The eNOS intron 4 polymorphism may be a marker of multi-vessel CAD in African Americans and Caucasians.

Adult↗
PubMed1989

Genetic cause of a juvenile form of Sandhoff disease. Abnormal splicing of beta-hexosaminidase beta chain gene transcript due to a point mutation within intron 12.

Abnormal beta-hexosaminidase beta chain cDNA clones were isolated from a library constructed from cultured fibroblasts of a patient with a juvenile form of Sandhoff disease (genetic beta-hexosaminidase A and B deficiency). Sequence analysis of a cDNA clone isolated from these fibroblasts contained an extra 24-base segment between exons 12 and 13. This segment was identified as the 3' terminus of intron 12. The remainder of the coding sequence was completely normal. The same 24-base insertion was found in four additional clones by sequencing. Restriction mapping analysis of seven other clones was consistent with the presence of the same 24-base intron 12 segment. This insertion is inframe and adds 8 amino acids between amino acids 491 and 492 of the primary sequence of the normal enzyme protein. It is located only 5 amino acids away from a possible glycosylation site. The finding is consistent with the slightly larger than normal size of the beta subunit precursor protein observed by immunoprecipitation. No normally spliced mRNA was detected. Gene amplification by the polymerase chain reaction and subsequent sequencing of genomic DNA indicated that the patient was a compound heterozygote. In one allele, there was a single nucleotide transition from normal G to A at 26 bases from the 3' terminus of intron 12. This mutation generates a consensus sequence for the 3' splice site for an intron, CAG/G, and thus explains the abnormal mRNAs that retain 24 bases of the 3' terminus of intron 12. The intron 12 and flanking exons 12 and 13 sequences were normal in the other allele, which is a priori also genetically abnormal. The other mutant allele therefore is likely to be of an mRNA-negative type.

Amino Acid Sequence↗
PubMed1988

The first intron of the alpha 1(I) collagen gene contains several transcriptional regulatory elements.

The first intron of the human alpha 1(I) collagen gene contains a negatively acting element that inhibits transcription of the chloramphenicol acetyltransferase gene driven by either a collagen or an SV40 basal promoter (Bornstein, P., McKay, J., Morishima, J., Devarayalu, S., and Gelinas, R. E. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, in press). We now find that this element is flanked by sequences that both neutralize the inhibitory effect and impart a net positive effect on transcription. A collagen-human growth hormone minigene was constructed in which varying lengths of the collagen intron were retained. Plasmids were transfected into chick tendon fibroblasts, and transcriptional activity was measured by solution hybridization with an antisense RNA probe. The presence of the intact intronic sequence stimulated transcription by a factor of 2-3-fold in comparison with intron-deleted plasmids. However, the isolated negatively acting element inhibited transcription by a factor of 15-20-fold. Surprisingly, this effect was markedly orientation-dependent. Intronic segments flanking the negatively acting element stimulated transcription both when cloned 5' to the collagen promoter in chloramphenicol acetyltransferase-based plasmids and 3' in collagen-human growth hormone constructions. We conclude that expression of the alpha 1(I) collagen gene is controlled by several intronic elements that function coordinately with 5'-flanking and promoter elements.

Acetyltransferases↗
PubMed1993

Identification of transcriptional cis-elements in introns 7 and 9 of the myeloperoxidase gene.

We studied the transcriptional cis-acting elements of the myeloperoxidase gene, which is expressed during the promyelocyte stage of granulocyte development by assay of transient expression of the chloramphenicol acetyltransferase (CAT) gene in myeloid leukemia SKM-1 cells and analysis of the DNA binding sites for HL-60 nuclear factors. Assay of CAT expression dependent on restriction fragments isolated from genomic clones indicated that the fragments located on introns 7 and 9 enhanced the expression. Methylation interference experiments showed that the guanine residues in a consensus sequence of an estrogen response element in the intron 7 fragment interacted with a nuclear factor. Gel retardation analysis indicated that this interaction of the intron 7 fragment with the nuclear factor was specifically inhibited by an oligodeoxynucleotide containing the 21-base pair (bp) estrogen response element. DNase I footprint analysis revealed that a 36-bp-specific sequence of the intron 9 fragment was protected from DNase I by nuclear extracts. This sequence contained a palindromic sequence consisting of the conserved half-motif of an estrogen response element with 5-bp spacing. The interaction of the intron 9 fragment with the nuclear extracts was specifically inhibited by an oligodeoxynucleotide of the 36-bp sequence. Furthermore, the 21- and 36-bp oligodeoxynucleotides in the constructs enhanced CAT expression in the cells. These results suggest that these elements in introns 7 and 9 are involved in expression of the myeloperoxidase gene.

Base Sequence↗
PubMed1993

Efficiency of introns from various origins in fish cells.

Several vectors containing (1) regulatory regions from Rous sarcoma virus (RSV), human cytomegalovirus (CMV), and herpes simplex thymidine kinase (TK); (2) introns from early or late SV40 genes and from trout growth hormone gene (tGH); (3) chloramphenicol acetyltransferase gene (CAT); and (4) transcription terminators from SV40 were transfected into carp EPC cells, salmon CHSE cells, tilapia TO2 cells, quail QT6 cells, and hamster CHO cells. CAT activity was measured in extracts from several cell lines 3 days after transfection and in the fish EPC stable clones. The CMV and RSV promoters were the most potent in all cell types. The intron from late SV40 genes (VP1 intron) worked properly in QT6 and CHO cells but not in EPC and very weakly in TO2 cells. The tGH intron was efficient in all cell types but preferentially in fish cells. The small t intron from SV40 was processed in all cell types. The small t and, to a lesser extent, the tGH introns amplified expression of cat gene in stable clones, in comparison to the transiently transfected cells. These results indicate that elements from mammalian genes may not be properly recognized by the fish cellular machinery and in an unpredictable manner. This finding suggests that vectors prepared to express foreign genes in transfected cultured fish cells and transgenic fish should preferably contain DNA sequences from fish genes or, alternatively, those sequences from mammalian genes that have been previously proved to be compatible with the fish cellular machinery.

Animals↗
PubMed1995

Secondary structure conservation of the U3 small nucleolar RNA introns in Saccharomyces.

Using a strategy based on PCR amplification of DNA and sequence analysis, we showed that the presence of introns with the characteristic features of introns spliced in a spliceosome, in the U3A and U3B snoRNA genes that code for the U3 small nucleolar RNA, is not a property restricted to Saccharomyces cerevisiae. It is probably an ancient property of yeasts from the genus Saccharomyces. We detected the U3A and U3B snoRNA genes in Saccharomyces bayanus and in a lager brewing yeast strain. The U3A and U3B intronic sequences are highly conserved. Two additional "U3B-like" snoRNA genes were detected in the lager brewing yeast. Their intronic sequences show several differences, when compared to the U3B intronic sequence. However, despite the numerous mutations, the intron secondary structure is conserved, especially, the central structure. This strongly suggests an important role of this central stem/loop structure for spliceosome assembly and efficient splicing.

Base Sequence↗
PubMed1996

A group I plant intron accumulates as circular RNA forms with extensive 5' deletions in vivo.

Analysis by a PAGE approach for detecting small circular RNAs showed the existence of one such molecular species (RNA 1) accumulating at high levels in cherimoya. Sequencing of cDNA clones of RNA 1 revealed a size of 281 nt and a sequence identical to the 3'-terminal region of the 494-nt tRNALeu(UAA) group I intron from cherimoya. Northern blot hybridizations with a probe complementary to RNA 1 showed that this RNA coexists in vivo with its corresponding linear form, with the presumed full-length intron, and with minor amounts of two additional small circular species (RNAs 2 and 3). RNAs 2 and 3 had sizes of 216 and 156 nt, respectively, and sequences identical to different moieties of the 3'-terminal region of the tRNALeu(UAA) intron. The three cyclization sites giving rise to RNAs 1, 2, and 3, located within loop 8, are preceded by CUU or UUU trinucleotides and followed by sequences capable of forming base pairing interactions with the internal guide sequence characteristic of group I introns. The good correlation observed between the stabilities of these interactions and the in vivo accumulation levels of the corresponding cherimoya circular RNAs support the hypothesis that they emerge through a common mechanism similar to that advanced previously for the generation of circular RNAs derived from other group I introns. The lack of interactions of similar stabilities in tobacco, in which no circular RNAs derived from the tRNALeu(UAA) intron were detected, is consistent with this proposal, although other factors are also probably important in the synthesis and accumulation of the small circular RNAs in cherimoya.

Base Sequence↗
PubMedDate not supplied

Intron 9 retention in gene transcripts suggests involvement of CD44 in the tumorigenesis of ovarian cancer.

Increased or de novo expression of certain CD44 variant isofoms as well as abnormal processing of pre-mRNA of the CD44 gene have been found in different malignant human tumors. Retention of the non-coding intron 9 sequence in CD44 mRNA especially, appears to be a discriminating aberrant splicing pattern in several cancer tissues and cells. We examined the expression of the CD44 intron 9 sequence in the mRNA of 45 permanent ovarian cancer cell lines and in normal ovarian tissue. Abnormal retention of intron 9 was found in 19 (60%) cell lines expressing CD44 std. Normal ovarian tissue as well as all ovarian cancer cells without CD44 s t d expression were found to be negative for intron 9 retention. Our results indicate that besides newly acquired expression of certain CD44 isoforms abnormal retention of the non-coding intron 9 sequence in CD44 gene transcripts is also a common and maybe crucial event in the tumorigenesis of ovarian cancer. Since intron 9 is not retained in normal ovarian tissue this aberration might serve as a marker for human adenocarcinoma of the ovary.

Adenocarcinoma↗
PubMed1986

The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located.

Amino Acid Sequence↗
PubMed1986

An intron in a ribosomal protein gene from Tetrahymena.

We have cloned and sequenced a single copy gene encoding a ribosomal protein from the ciliate Tetrahymena thermophila. The gene product was identified as ribosomal protein S25 by comparison of the migration in two-dimensional polyacrylamide gels of the protein synthesized by translation in vitro of hybrid-selected mRNA and authentic ribosomal proteins. The proteins show strong homology to ribosomal protein S12 from Escherichia coli. The coding region of the gene is interrupted by a 979-bp intron 68 bp downstream of the translation start. This is the first intron in a protein encoding gene of a ciliate to be described at the nucleotide sequence level. The intron obeys the GT/AG rule for splice junctions of nuclear mRNA introns from higher eukaryotes but lacks the pyrimidine stretch usually found in the immediate vicinity of the 3' splice junction. The structure of the intron and the fact that it is found together with the well described self-splicing rRNA intron is discussed in relation to the evolution of RNA splicing.

Journal Article↗
PubMed1984

Human dihydrofolate reductase gene organization. Extensive conservation of the G + C-rich 5' non-coding sequence and strong intron size divergence from homologous mammalian genes.

The complete human dihydrofolate reductase (DHFR) gene has been cloned from four recombinant lambda libraries constructed with the DNA from a methotrexate-resistant human cell line with amplified DHFR genes. The detailed organization of the gene has been determined by restriction mapping of the cloned fragments and DNA sequencing of all the protein coding regions and adjacent intron segments, and shown to correspond to that of the native human DHFR gene. The gene spans a length of approximately 29 X 10(3) bases from the ATG initiator codon to the end of the 3' untranslated region, and contains five introns that interrupt the protein coding sequence. The number and positions of introns are identical to those found in the mouse gene. By contrast, the size of the homologous introns (with the exception of the first one) varies greatly, up to several fold, in the genes from man, mouse and Chinese hamster; the intron sequences also exhibit a great divergence, except in the junction regions. A striking sequence homology, extending over several hundred nucleotides, exists between the human and mouse gene 5' non-coding regions. These regions are characterized by an unusually high G + C content, 72% and 66% in the human and mouse genes, respectively, which is maintained in the first coding segment and first intron, and is in sharp contrast to the relatively low G + C content (approximately 40%) of the remainder of the gene.

Animals↗