SEARCH · PubMed Health

Results for “Introns”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 721 records · Page 40Linked to original sources

PubMed2026

Enhancing CRISPR-Cas12a base editing in plants with LbCas12a variants and introns.

Cytosine base editors (CBEs) and adenine base editors (ABEs) are powerful tools for precise genome editing in plants. Conventionally, such base editors are built upon the CRISPR-Cas9 systems where Cas9 nickases are used. To expand the base editing scope and minimize off-target effects, base editors derived from the CRISPR-Cas12a systems are desired. However, the use of deactivated Cas12a (dCas12a) in such base editors constrains the editing activity, preventing the wide use of Cas12a base editors for plant research and trait development. In this study, we demonstrate the use of an ABE based on the efficient LbCas12a-RRV variant to introduce herbicide-resistant mutations in OsACCase in rice. To improve Cas12a CBEs and ABEs, we inserted introns into the coding sequence of dLbCas12a-RRV. This intron-containing Cas12a-CBE shows substantial improvement in editing efficiency in rice, compared to the intron-less counterparts. By contrast, the improvement of ABE with the intron-containing dLbCas12a-RRV is very limited, partly due to the already high baseline editing efficiency of the intron-less dLbCas12a-RRV ABE. Testing of these base editors in poplar shows elevated C-to-T base editing by dLbCas12a-RRV-intron-CBE. For A-to-G editing, ABEs built upon dLbCas12a-RV and dLbCas12a-RRV variants showed significant improvement over ABEs derived from wild-type LbCas12a and the ttLbCas12a variant. The addition of introns to dLbCas12a-RRV does not further improve the base editing efficiency. With whole genome sequencing in rice, we evaluated genome editing specificities with these improved Cas12a base editors. Our analyses show that both intron-containing Cas12a CBE and ABE barely introduce guide RNA-dependent off-target mutations. However, they can generate guide RNA-independent off-target mutations, which are likely attributed to the high enzymatic activities of the deaminases. Collectively, our study demonstrates the successful use of a Cas12a base editor for trait development and reports improved Cas12a CBEs and ABEs for precise base editing in plants.

Oryza↗

PubMed2000

Group II introns designed to insert into therapeutically relevant DNA target sites in human cells.

Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy.

Base Pairing↗

PubMed1998

Sequence variation of the tRNA(Leu) intron as a marker for genetic diversity and specificity of symbiotic cyanobacteria in some lichens.

We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNA(Leu) (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNA(Leu) (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNA(Leu) (UAA) intron can be of great value when examining cyanobacterial diversity.

Base Sequence↗

PubMed1987

Interrupted thymidylate synthase gene of bacteriophages T2 and T6 and other potential self-splicing introns in the T-even bacteriophages.

Southern hybridization analyses of procaryotic DNA from Escherichia coli, lambda bacteriophage, and T1 to T7 phages were carried out. The hybridization probes used consisted of DNA restriction fragments derived from the T4 phage intron-containing thymidylate synthase gene (td) and short synthetic oligodeoxynucleotides defining specific exon and intron regions of the gene. It was shown that intact as well as restricted DNA from the T-even phages hybridized not only to both T4 phage td intron- and exon-specific probes but also to probes defining the td 5' (exon I-intron) and 3' (intron-exon II) presplice junctions. These data strongly suggest that, analogous to the T4 phage, only the T2 and T6 phages among the procaryotes tested contain interrupted td genes. The td intervening sequence in each phage is roughly 1 kilobase pair (kb) in size and interrupts the td gene at a site analogous to that in the T4 phage. This was confirmed by data from Northern (RNA) hybridization analysis of td-specific in vitro transcripts of these phage DNAs. [alpha-32P]GTP in vitro labeling of total RNA from T4 phage-infected cells produced five species of labeled RNAs that were 1, 0.9, 0.83, 0.75, and 0.6 kb in size. Only the 1-, 0.9-, and 0.75-kb species were labeled in RNA from T2- or T6-infected cells. The commonly present 1-kb RNA is the excised td intron, which exists in both linear and circular forms in the respective T-even-phage-infected cells, while the 0.6-kb RNA unique to T4 may be the excised intron derived from the ribonucleotide reductase small subunit gene (nrdB) of the phage. The remaining labeled RNA species are likely candidates for other self-splicing introns.

DNA, Bacterial↗

PubMed2005

Unusual group II introns in bacteria of the Bacillus cereus group.

A combination of sequence and structure analysis and reverse transcriptase PCR experiments was used to characterize the group II introns in the complete genomes of two strains of the pathogen Bacillus cereus. While B. cereus ATCC 14579 harbors a single intron element in the chromosome, B. cereus ATCC 10987 contains three introns in the chromosome and four in its 208-kb pBc10987 plasmid. The most striking finding is the presence in B. cereus ATCC 10987 of an intron [B.c.I2(a)] located on the reverse strand of a gene encoding a putative cell surface protein which appears to be correlated to strains of clinical origin. Because of the opposite orientation of B.c.I2(a), the gene is disrupted. Even more striking is that B.c.I2(a) splices out of an RNA transcript corresponding to the opposite DNA strand. All other intragenic introns studied here are inserted in the same orientation as their host genes and splice out of the mRNA in vivo, setting the flanking exons in frame. Noticeably, B.c.I3 in B. cereus ATCC 10987 represents the first example of a group II intron entirely included within a conserved replication gene, namely, the alpha subunit of DNA polymerase III. Another striking finding is that the observed 3' splice site of B.c.I4 occurs 56 bp after the predicted end of the intron. This apparently unusual splicing mechanism may be related to structural irregularities in the 3' terminus. Finally, we also show that the intergenic introns of B. cereus ATCC 10987 are transcribed with their upstream genes and do splice in vivo.

Bacillus cereus↗

PubMed1991

A generic intron increases gene expression in transgenic mice.

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

Animals↗

PubMed2003

Cotranscriptional recruitment of the U1 snRNP to intron-containing genes in yeast.

Evidence that pre-mRNA processing events are temporally and, in some cases, mechanistically coupled to transcription has led to the proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. Here we address two key questions raised by this proposal: (i) whether the U1 snRNP, which binds to the 5' splice site of each intron, is recruited cotranscriptionally in vivo and, (ii) if so, where along the length of active genes the U1 snRNP is concentrated. Using chromatin immunoprecipitation (ChIP) in yeast, we show that elevated levels of the U1 snRNP were specifically detected in gene regions containing introns and downstream of introns but not along the length of intronless genes. In contrast to capping enzymes, which bind directly to Pol II, the U1 snRNP was poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP was dependent on RNA synthesis and was abolished by intron removal. Microarray analysis revealed that intron-containing genes were preferentially selected by ChIP with the U1 snRNP. Thus, U1 snRNP accumulation at genes correlated with the presence and position of introns, indicating that introns are necessary for cotranscriptional U1 snRNP recruitment and/or retention.

Chromatin↗

PubMed1986

Anomalous placement of introns in a member of the intermediate filament multigene family: an evolutionary conundrum.

The origin of introns and their role (if any) in gene expression, in the evolution of the genome, and in the generation of new expressed sequences are issues that are understood poorly, if at all. Multigene families provide a favorable opportunity for examining the evolutionary history of introns because it is possible to identify changes in intron placement and content since the divergence of family members from a common ancestral sequence. Here we report the complete sequence of the gene encoding the 68-kilodalton (kDa) neurofilament protein; the gene is a member of the intermediate filament multigene family that diverged over 600 million years ago. Five other members of this family (desmin, vimentin, glial fibrillary acidic protein, and type I and type II keratins) are encoded by genes with six or more introns at homologous positions. To our surprise, the number and placement of introns in the 68-kDa neurofilament protein gene were completely anomalous, with only three introns, none of which corresponded in position to introns in any characterized intermediate filament gene. This finding was all the more unexpected because comparative amino acid sequence data suggest a closer relationship of the 68-kDa neurofilament protein to desmin, vimentin, and glial fibrillary acidic protein than between any of these three proteins and the keratins. It appears likely that an mRNA-mediated transposition event was involved in the evolution of the 68-kDa neurofilament protein gene and that subsequent events led to the acquisition of at least two of the three introns present in the contemporary sequence.

Amino Acid Sequence↗

PubMed1988

Pseudouridine modification in the tRNA(Tyr) anticodon is dependent on the presence, but independent of the size and sequence, of the intron in eucaryotic tRNA(Tyr) genes.

In Saccharomyces cerevisiae, pseudouridine formation in the middle position of the tRNA(Tyr) anticodon (psi 35) is dependent on the presence of the intron in the tRNA(Tyr) gene (Johnson and Abelson, Nature 302:681-687, 1983). Drosophila melanogaster tRNA(Tyr) genes contain introns of three size classes: 20 or 21 base pairs (bp) (six genes), 48 bp (one gene), and 113 bp (one gene). As in yeast, removal of the intron led to loss of psi 35 in the anticodon when transcription was assayed in Xenopus laevis oocytes. All Drosophila intron sizes supported psi 35 formation. The same results were obtained with the homologous X. laevis tRNA(Tyr) genes containing introns of 12 or 13 bp or with a deleted intron. The introns of yeast (Nishikura and DeRobertis, J. Mol. Biol. 145:405-420, 1981), D. melanogaster, and X. laevis tRNA(Tyr) wild-type genes, while they all supported psi 35 synthesis, did not share any consensus sequences. As discussed, these results, taken together, suggest that for appropriate function the psi 35 enzyme in the X. laevis oocyte needs the presence of an unqualified intron in the tRNA gene and a tRNA(Tyr)-like structure in the unprocessed tRNA precursor.

Animals↗

PubMed2006

Detection of potentially valuable polymorphisms in four group I intron insertion sites at the 3'-end of the LSU rDNA genes in biocontrol isolates of Metarhizium anisopliae.

BACKGROUND: The entomopathogenic anamorphic fungus Metarhizum anisopliae is currently used as a biocontrol agent (BCA) of insects. In the present work, we analyzed the sequence data obtained from group I introns in the large subunit (LSU) of rDNA genes with a view to determining the genetic diversity present in an autochthonous collection of twenty-six M. anisopliae isolates selected as BCAs. RESULTS: DNA fragments corresponding to the 3'-end of the nuclear LSU rDNA genes of 26 M. anisopliae isolates were amplified by PCR. The amplicon sizes ranged from 0.8 to 3.4-kb. Four intron insertion sites, according to Escherichia coli J01695 numbering, were detected--Ec1921, Ec2066, Ec2449 and Ec2563--after sequencing and analysis of the PCR products. The presence/absence of introns allowed the 26 isolates to be distributed into seven genotypes. Nine of the isolates tested showed no introns, 4 had only one, 3 two, and 10 displayed three introns. The most frequent insertion sites were Ec1921 and Ec2449. Of the 26 isolates, 11 showed insertions at Ec2563 and a 1754-bp sequence was observed in ten of them. The most-parsimonious (MP) tree obtained from parsimony analysis of the introns revealed a main set containing four-groups that corresponded to the four insertion sites. CONCLUSION: Four insertion sites of group I introns in the LSU rDNA genes allowed the establishment of seven genotypes among the twenty-six biocontrol isolates of M. anisopliae. Intron insertions at the Ec2563 site were observed for first time in this species.

3' Untranslated Regions↗

PubMedDate not supplied

Divergence between genotype and phenotype in relatives of patients with the intron 2 mutation of steroid-21-hydroxylase.

We studied 95 patients and their relatives with the classical salt wasting (SW) and simple virilizing (SV) form of CAH. SSCP/heteroduplex analysis allowed fast and efficient screening for the most common 21-hydroxylase mutations (e.g. deletions, splice site mutation in intron 2 (bp 656), Ile172Asn mutation in exon 4) and determination of the relative intensities of CYP21A and CYP21B genes. The splice site mutation in intron 2 was found as the most frequent cause of 21-hydroxylase deficiency (35% of our patients). There is a strong genetic association between the mutation in intron 2 and the SW form of CAH. On the other hand, about 20% of our patients with the intron 2 mutation have the SV phenotype. Interestingly, homozygous splice site mutations in intron 2 were also detected in some parents or other relatives with no phenotypic changes typical for CAH (clinical evaluation, steroid hormone levels). In those patients with SV-CAH and especially in the relatives with the homozygous intron 2 mutation and an unaffected phenotype, the splice site mutation could be "leaky". mRNA-splicing in the adrenal cortex should result in a high degree of normal mRNA species. This is in contrast to in vitro expression studies of CYP21B genes containing the intron 2 mutation, performed by other groups. However, the results of in vitro expression studies are not always reflecting the in vivo conditions in the adrenal cortex. This situation is in good agreement with the variable degree of normal spliced mRNA and different phenotypic severity in intron mutations found in thalassemia.

Adrenal Hyperplasia, Congenital↗

PubMed1999

Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements.

To investigate mechanisms of human angiotensin II type 2 receptor (hAT2) gene regulation we functionally characterized the promoter and downstream regions of the gene. 5'-Terminal deletion mutants from -1417/+100 to -46/+100 elicited significant but low functional activity in luciferase reporter gene assays with PC12W cells. Inclusion into the promoter constructs of intron 1 and the transcribed region of the hAT2 gene up to the translation start enhanced luciferase activity 6.7+/-1.6-fold and 11.6+/-1.7-fold (means+/-S.E.M.) respectively, whereas fusion of the promoter to the spliced 5' untranslated region of hAT2 cDNA did not, which indicated an enhancement caused by intronic sequence elements. Reverse transcriptase-mediated PCR confirmed that the chimaeric hAT2-luciferase mRNA was regularly spliced in PC12W cells. A Northern blot analysis of transfected cells showed levels of luciferase mRNA expression consistent with the respective enzyme activities. Mapping of intron 1 revealed that a 12 bp sequence in the centre of the intron was required for the increase in promoter activity, whereas the 5' adjacent intronic region mediated a decrease in luciferase activity. Mutation of the 12 bp region led to altered protein binding and markedly decreased luciferase activity. Cloned into a promoterless luciferase vector, a 123 bp intron 1 fragment was able to direct reporter gene expression to the same activity as occurred in conjunction with the 5' flanking region. These results indicate that sequence elements in intron 1 are necessary for efficient transcription of hAT2. In reporter gene assays, intron 1 might by itself function as a promoter and initiate transcription from an alternative start point.

5' Untranslated Regions↗

PubMed1999

[The sequence analysis of the 3' end of intron 15 from human LDL receptor gene and its application in the study of gene diagnosis for familial hypercholesterolaemia].

To understand intron 15 of human LDL receptor gene, the DNA fragments from exon 15 to exon 16 and the 3' end of intron 15 were amplified with long chain PCR and anchored PCR. The 3' end of intron 15 was sequenced with Dynalbeads-Streptavidin Solid Phase technique. The sequence analysis showed that the 3' end of intron 15 contained the 3' splicing site and the branch site at 31 nucleotides upstream of the 3' end. Besides the authentic branch site, it is possible that the 3' end of intron 15 contains a cryptic site (GCCTCAC) at 20 nucleotides upstream of the 3' end. The sequences suggest that the PvuII polymorphism at Intron 15 is caused by the T-C substitution. According to the sequences of the 3' end, the new PCR-RFLP protocol for detection of PvuII polymorphism at intron 15 was developed. Using this protocol a representative familial hypercholesterolaemia family was identified with linkage analysis of PvuII polymorphism at intron 15.

Base Sequence↗

PubMed2003

Modification of pGH cDNA using the first intron and adenovirus-mediated expression in CHO cells.

OBJECTIVE: This study was conducted to investigate the function of the first intron of porcine growth hormone (pGH) gene in the gene expression. METHODS: PCR method was used to amplify the first intron from pig genomic DNA. The intron was then inserted into pGH cDNA to construct pGH cDNA-intron (pGH cDNA-in). The recombinant adenoviruses containing pGH cDNA and pGH cDNA-in genes under control of CMV promoter were generated by homologous recombination method in HEK 293 cells respectively. The effect of the first intron on gene expression was evaluated by comparing the expression levels of pGH cDNA-in and pGH cDNA mediated by adenovirus vectors in CHO cells. RESULTS: The expression level of pGH cDNA containing the first intron increased by 117%, which was significantly higher than that of pGH cDNA without the intron (P < 0.001). CONCLUSION: The first intron of pGH gene has the function to improve pGH gene expression.

Adenoviridae↗

PubMed2004

Distributions of exons and introns in the human genome.

The human genome is revisited using exon and intron distribution profiles. The 26,564 annotated genes in the human genome (build October, 2003) contain 233,785 exons and 207,344 introns. On average, there are 8.8 exons and 7.8 introns per gene. About 80% of the exons on each chromosome are < 200 bp in length. < 0.01% of the introns are < 20 bp in length and < 10% of introns are more than 11,000 bp in length. These results suggest constraints on the splicing machinery to splice out very long or very short introns and provide insight to optimal intron length selection. Interestingly, the total length in introns and intergenic DNA on each chromosome is significantly correlated to the determined chromosome size with a coefficient of correlation r = 0.95 and r = 0.97, respectively. These results suggest their implication in genome design.

Alternative Splicing↗

PubMed2006

First exons and introns--a survey of GC content and gene structure in the human genome.

Most transcriptional regulatory elements are located in non-coding DNA. In particular, some first introns play a vital role in transcriptional control and splicing. The length and GC-content of first exons and introns in complex organisms suggests that these structural units are likely to be important functional elements in large genomes. Hence, in this paper we perform a systematic comparison of exon-intron structure and GC content on all known genes in the human genome. Our in-silico analysis found that the GC content of introns and exons varies significantly depending on their length. On average, the first intron of a gene is significantly longer than other introns in the same gene. Our results also show that first introns and exons are more GC rich than last and internal. This study provides insight into the structure of eukaryotic genes. These results confirm and expand the previously identified regulatory potential of first exons and introns.

Base Composition↗

PubMed1996

Glycogenosis type VII (Tarui disease) in a Swedish family: two novel mutations in muscle phosphofructokinase gene (PFK-M) resulting in intron retentions.

Phosphofructokinase (PFK) plays a major role in glycolysis. Human PFK is composed of three isoenzyme subunits (muscle [Ml, liver [L], and platelet [P]), which are encoded by different genes. Deficiency of muscle isoenzyme (PFK-M), glycogenosis type VII (Tarui disease), is an autosomal recessive disorder characterized by an exertional myopathy and hemolytic syndrome. Several disease-causing mutations have been identified in the PFK-M gene in Japanese, Ashkenazi Jewish, Italian, French Canadian, and Swiss patients. We describe the genetic defect in a Swedish family with affected individuals in two generations. The patients are compound heterozygotes: two different mutations result in retention of intron 13 or intron 16 sequences into mRNA. A G1127A transition destroys the 5' donor site of intron 13, resulting in a 155-nt retention of the intronic sequence. An a-to-g base change in intron 16 creates a new acceptor splice site, resulting in a 63-nt retention of intronic sequence. Both mutations are predicted to result in premature termination of translation. Some of the transcripts generated from the intron 16 mutated allele also contain intron 10 sequence unspliced.

Adolescent↗

PubMed1998

Human U19 intron-encoded snoRNA is processed from a long primary transcript that possesses little potential for protein coding.

While exons were originally defined as coding regions of split eukaryotic genes, introns have long been considered as mainly noncoding "genetic junk." However, recognition that a large number of small nucleolar RNAs (snoRNAs) are processed from introns of pre-mRNAs demonstrated that introns may also code for functional RNAs. Moreover, recent characterization of the mammalian UHG gene that encodes eight box C/D intronic snoRNAs suggested that some genes generate functional RNA products exclusively from their intron regions. In this study, we show that the human U19 box H/ACA snoRNA, which is encoded within the second intron of the U19H gene, represents the only functional RNA product generated from the long U19H primary transcript. Splicing of the U19H transcript, instead of giving rise to a defined RNA, produces a population of diverse U19H RNA molecules. Although the first three exons of the U19H gene are preserved in each processed U19H RNA, the 3' half of the RNA is generated by a series of apparently random splicing events. Because the U19H RNA possesses limited potential for protein coding and shows a predominant nucleoplasmic localization, we suggest that the sole function of the U19H gene is to express the U19 intronic snoRNA. This suggests that, in marked contrast to our previous dogmatic view, genes generating functionally important RNAs exclusively from their intron regions are probably more frequent than has been anticipated.

Alternative Splicing↗